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    • 专利摘要:
    A method for producing a sugar liquid and apparatus The present invention is intended to provide a method of cellulosic hydrolysis using a filamentous fungus derived cellulase as a carbohydrase, which method comprises the step of adding carbohydrase to cellulose to perform primary hydrolysis and then subjecting the primary hydrolyzate to solid-liquid separation into a primary sugar liquid and solids; the step of adding water to the solids and performing secondary hydrolysis, followed by subjecting the secondary hydrolyzate to solid-liquid separation into a secondary sugar liquid and a residue; and the step of filtering the primary sugar liquid and / or secondary sugar liquid through an ultrafiltration membrane, and recovering the carbohydrate from the feed side and recovering a permeate side sugar solution. Thus, a method for reducing the amount of enzyme such as cellulase used in a method for producing a sugar liquid from the pretreated cellulose is provided.
    公开号:BR112012023159B1
    申请号:R112012023159-6
    申请日:2011-03-14
    公开日:2019-05-07
    发明作者:Hiroyuki Kurihara；Atsushi Minamino；Katsushige Yamada；Yuki Yamamoto
    申请人:Toray Industries, Inc.；
    IPC主号:
    专利说明:

    "METHOD TO PRODUCE A SUGAR LIQUID"
    Field of the Invention [001] The present invention relates to a method for producing a sugar liquid from cellulose, and an apparatus for the method.
    Background of the Invention [002] Fermentation processes to produce chemicals that use sugars as raw materials have been used to produce various industrial materials. Currently, as sugars to be used as raw materials for fermentation, sugars derived from food materials, such as sugar cane, starch and sugar beet are used industrially. However, as an increase in the price of food materials is expected due to the future increase in the world population, or in an ethical view, due to the fact that sugars as industrial materials can compete with sugars for food, it is necessary to design a process in the future that efficiently produces a sugar liquid from a renewable non-food resource, that is, a biomass containing cellulose, or a process that uses a sugar liquid obtained as a raw material for fermentation to efficiently convert the sugar liquid into an industrial material.
    [003] Examples of the methods disclosed for producing a sugar liquid from a cellulose-containing biomass include methods for producing sugar liquids by the acid hydrolysis of cellulose and hemicellulose using concentrated sulfuric acid (Patent Document 1 and 2) and a method in which a cellulose-containing biomass is subjected to hydrolysis treatment using diluted sulfuric acid and then is treated enzymatically with cellulase or similar to produce a sugar liquid (Non-Patent Document 1). In addition, examples of disclosed methods that do not use acid include a method in which a biomass
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    2/85 containing cellulose is hydrolyzed using subcritical water at about 250 ° C to 500 ° C to produce a sugar liquid (Patent Document 3), a method in which a cellulose-containing biomass is subjected to subcritical water treatment and then is treated enzymatically to produce a sugar liquid (Patent Document 4), and a method in which a cellulose-containing biomass is subjected to hydrolysis treatment with pressurized hot water at 240 ° C to 280 ° C and then is treated enzymatically to produce a liquid sugar (Patent Document 5).
    [004] In recent years, methods of hydrolysis of a biomass that use less energy and generate less environmental burden, but produce sugar with high yields have been studied extensively. However, these methods using enzymes have the inconvenience of high enzyme costs.
    [005] To solve these technical problems, methods have been suggested for recovery and reuse of enzymes used in hydrolysis. Examples of these disclosed methods include a method in which continuous solid-liquid separation is performed with a spin filter and the sugar liquid obtained is filtered through an ultrafiltration membrane to recover the enzymes (Patent Document 6), a method in which a surfactant is fed in the enzymatic saccharification stage, to suppress enzyme adsorption and increase recovery efficiency (Patent Document 7), a method in which the residue produced by enzymatic saccharification is subjected to an electrical treatment to recover the enzymatic component (Patent Document 8) and a method in which the residue produced by enzymatic saccharification is fed back into another batch of biomass and with that the enzymes are reused (Patent Document 9).
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    3/85
    Prior Art Documents
    Patent Document
    Patent Document 1: PCT Patent Application Translated from
    Japan Open to Public Inspection No. 11-506934
    Patent Document 2: JP 2005-229821 A
    Patent Document 3: JP 2003-212888 A
    Patent Document 4: JP 2001-95597 A
    Patent Document 5: JP 3041380 B
    Patent Document 6: JP 2006-87319 A
    Patent Document 7: JP 63-87994 A
    Patent Document 8: JP 2008-206484 A
    Patent Document 9: JP 55-144885 A
    Non-Patent Documents
    Non-Patent Document 1: A. Aden et al. “Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover” NREL Technical Report (2002)
    SUMMARY OF THE INVENTION
    Problems That Will Be Solved by the Invention [006] Methods for producing sugar liquids by recovering / reusing the enzyme have been developed as described above, but the effects of these methods have been insufficient in view of the reduction in the amount of the enzyme used. Therefore, the present invention seeks to develop a process in which the effect of reducing the amount of enzyme is greater than the effects in conventional methods.
    Means to Solve the Problems [007] The present inventors studied intensively to solve the aforementioned problems, and, consequently, a method was invented
    Petition 870190015245, of 02/14/2019, p. 10/177
    4/85 of cellulose hydrolysis using a cellulase derived from filamentous fungus as a carbohydrase, a method that comprises: the step of adding cellulose to carbohydrase to perform primary hydrolysis and then subject the primary hydrolyzate to solid-liquid separation in a primary sugar liquid and solids; the step of adding water to the solids and performing secondary hydrolysis, followed by subjecting the secondary hydrolyzate to solid-liquid separation in a secondary sugar liquid and a residue; and the step of filtering the primary sugar liquid and / or secondary sugar liquid through an ultrafiltration membrane and recovering the carbohydrate from the feed side and recovering a sugar solution from the permeate side.
    [008] That is, the present invention has the numbers (1) to (13) below as constituents.
    [009] (1) A method for producing a sugar liquid using a filamentous fungus-derived cellulase as a carbohydrate to hydrolyze cellulose, the method comprising:
    the step of adding said carbohydrase to cellulose to carry out primary hydrolysis and then subject the primary hydrolyzate to solid-liquid separation in a liquid of primary sugar and solids;
    the step of adding water to the solids and performing secondary hydrolysis, followed by subjecting the secondary hydrolyzate to solid-liquid separation in a secondary sugar liquid and a residue; and the step of filtering the primary sugar liquid and / or secondary sugar liquid through an ultrafiltration membrane, and recovering the carbohydrate from the feed side and recovering a sugar liquid from the permeate side.
    [010] (2) The method for producing a sugar liquid according to (1), wherein the cellulase derived from filamentous fungus is cellulase derived from Trichoderma.
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    5/85 [011] (3) The method for producing a sugar liquid according to (1) or (2), in which the cellulose is derived from a processed product prepared by treatment with ammonia, hydrothermal treatment or biomass treatment with diluted sulfuric acid.
    [012] (4) The method for producing a sugar liquid according to any one of (1) to (3), wherein the secondary hydrolysis is hydrolysis in the presence of one or more selected from the group consisting of salts inorganic (except calcium salts), hydrophilic organic solvents, amino acids and non-ionic surfactants, and sugar liquids comprising such substances.
    [013] (5) The method for producing a sugar liquid according to (4), in which the inorganic salt (s) (except calcium salts) is / are one or more selected from from the group consisting of sodium salts, potassium salts, magnesium salts, sulfuric acid salts, ammonium salts, hydrochloric acid salts, phosphoric acid salts, acetic acid salts and nitric acid salts.
    [014] (6) The method for producing a sugar liquid according to (5), in which the inorganic salt (s) (except calcium salts) is / are one or more selected from of the group consisting of sodium chloride, sodium acetate, sodium sulfate, sodium hydrogen sulfate, sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium chloride, ammonium chloride, dipotassium hydrogen phosphate, ammonium sulphate, magnesium chloride and sulfate magnesium.
    [015] (7) The method for producing a sugar liquid according to (4), wherein the hydrophilic organic solvent (s) is / are one or more selected from the group which consists of methanol, ethanol, 1propanol, isopropanol, N, N-dimethylformamide, butanol, acetone, acetonitrile, ethylene glycol and glycerin.
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    6/85 [016] (8) The method for producing a sugar liquid according to (4), wherein the amino acid (s) is / are one or more selected from the group consisting of arginine, cysteine, glutamic acid, histidine and lysine.
    [017] (9) The method for producing a sugar liquid according to any one of (1) to (8), wherein the solid-liquid separation of a primary hydrolyzate and / or secondary hydrolyzate is filtration using a filter press.
    [018] (10) The method for producing a sugar liquid according to any one of (1) to (9), the method comprising the step of filtering the sugar liquid through a reverse osmosis membrane and / or membrane nanofiltration to concentrate the sugar liquid.
    [019] (11) An apparatus for the method for producing a sugar liquid according to any one of (1) to (10), the apparatus comprising as a stirring tank for primary hydrolysis; solid-liquid separation device; secondary hydrolysis tank or filtration device using filter press for secondary hydrolysis; solid-liquid separation device (s) for the primary hydrolyzate and / or secondary hydrolyzate; and an ultrafiltration membrane device for separating the carbohydrase and the sugar liquid from the primary sugar liquid and / or the secondary sugar liquid.
    [020] (12) An apparatus for the method for producing a sugar liquid according to any one of (1) to (10), the apparatus comprising as a constituent a reaction vessel for primary hydrolysis; filtration device using filter press having a warm water supply tank; circulation line to circulate the filtrate from filtration device using filter press for the tank of warm water supply; and
    Petition 870190015245, of 02/14/2019, p. 10/20
    7/85 ultrafiltration membrane device for separating the carbohydrase and the sugar liquid from the primary sugar liquid and / or the secondary sugar liquid.
    [021] (13) The apparatus according to (11) or (12), which comprises as a constituent a sugar liquid concentration device equipped with a reverse osmosis membrane and / or a nanofiltration membrane to concentrate the liquid of sugar obtained with the ultrafiltration membrane device.
    Effect of the Invention [022] In the present invention, primary hydrolysis is succeeded by solid-liquid separation, and the residual components of the enzyme contained in the obtained solids are used to carry out the secondary hydrolysis. This produces 1) the effect of increasing sugar yield and 2) the effect of increasing the amount of enzyme recovered. Therefore, the present invention is economically advantageous over conventional techniques.
    Brief Description of the Drawings [023] Figure 1 is a schematic diagram showing an embodiment of the method for producing a sugar liquid of the present invention.
    [024] Figure 2 is a schematic diagram showing an embodiment of the apparatus for carrying out the method for producing a sugar liquid of the present invention.
    [025] Figure 3 is a schematic diagram showing an embodiment of the apparatus for carrying out the method for producing a sugar liquid of the present invention.
    [026] Figure 4 is a schematic diagram showing an embodiment in which secondary hydrolysis in the method for producing a sugar liquid of the present invention is carried out in a filtration chamber using a filter press.
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    8/85 [027] Figure 5 is a schematic diagram showing an embodiment of the apparatus for carrying out the method for producing a sugar liquid of the present invention.
    [028] Figure 6 is a schematic diagram showing an embodiment of the apparatus for carrying out the method for producing a sugar liquid of the present invention.
    [029] Figure 7 is a schematic diagram showing an embodiment in which secondary hydrolysis in the method for producing a sugar liquid of the present invention is carried out in a filtration chamber using a filter press.
    [030] Figure 8 is a schematic diagram showing an embodiment of the apparatus for carrying out the method for producing a sugar liquid of the present invention.
    [031] Figure 9 is a diagram showing the results of the analysis of an enzyme contained in the secondary sugar liquid obtained by the method for producing a sugar liquid of the present invention.
    [032] Figure 10 is a schematic diagram showing an embodiment in which the secondary hydrolysis in the method for producing a sugar liquid of the present invention is carried out in a secondary hydrolysis tank that is separated from the tank for primary hydrolysis.
    [033] Figure 11 is a schematic diagram showing an embodiment in which the secondary hydrolysis in the method for producing a sugar liquid of the present invention is carried out in a secondary hydrolysis tank that is separated from the tank for primary hydrolysis.
    [034] Figure 12 is a schematic diagram showing an embodiment in which secondary hydrolysis in the method for producing a sugar liquid of the present invention is carried out in a secondary hydrolysis tank that is separated from the tank for primary hydrolysis.
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    9/85 [035] Figure 13 is a schematic diagram showing an embodiment in which the primary hydrolysis and the secondary hydrolysis in the method for producing a sugar liquid of the present invention are carried out in the same tank.
    [036] Figure 14 is a schematic diagram showing an embodiment in which a microfiltration membrane device is placed upstream of an ultrafiltration membrane device.
    [037] Figure 15 is a schematic diagram showing a modality in which cross-flow filtration is performed using a microfiltration membrane module.
    [038] Figure 16 is a schematic diagram showing a modality in which "dead end" filtration is performed using a module microfiltration membrane.
    Best Mode of Execution of the Invention [039] Expressive volumes of celluloses are contained in herbaceous biomasses such as bagasse, switchgrass grass, napier grass, Erianthus, corn remains, rice straw and wheat straw; and woody biomasses such as trees and construction waste materials. Biomasses that include cellulose can preferably be used as raw materials in the present invention.
    [040] Cellulose-containing biomass contains, in addition to cellulose and hemicellulose (hereinafter referred to as “cellulose” as a generic term for cellulose and hemicellulose), lignin and other similar substances that are aromatic macromolecules. Therefore, in cases where cellulose derived from a biomass is used as a raw material for a sugar liquid in the method of producing a sugar liquid of the present invention, the efficiency of enzymatic hydrolysis can be increased through pretreatment. Examples of the pre-treatment method for a biomass that
    Petition 870190015245, of 02/14/2019, p. 10/23
    10/85 contains cellulose include acid treatment, sulfuric acid treatment, diluted sulfuric acid treatment, alkaline treatment, lye treatment, ammonia treatment, hydrothermal treatment, subcritical water treatment, spray treatment and steam treatment. In the present invention, the pretreatment method is preferably ammonia treatment, hydrothermal treatment or treatment with dilute sulfuric acid.
    [041] Treatment with ammonia is carried out according to JP 2008-161125 A and JP 2008-535664 A. For example, ammonia is added to biomass in a concentration in the range of 0.1 to 15% by weight, and the treatment is carried out at 4 to 200 ° C, preferably 90 to 150 ° C. The ammonia to be added may be in a liquid or gaseous state. Additionally, the form of ammonia that will be added can be pure ammonia or aqueous ammonia. The number of times of treatment is not restricted, and the treatment can be performed 1 or more times. In particular, in cases where the treatment is carried out 2 or more times, the conditions for the first treatment may be different from the conditions for the second and subsequent treatment. The treated product obtained by treatment with ammonia needs to be subjected to ammonia neutralization or ammonia removal in order to carry out the enzymatic hydrolysis reaction. The neutralization of ammonia can be carried out after removing the solids from the hydrolyzate through solid-liquid separation or in the state in which the solids are contained. The acid reagent to be used for neutralization is not restricted. Ammonia can be removed by keeping the product treated with ammonia under reduced pressure to allow the ammonia to evaporate to the gaseous state. The removed ammonia can be recovered and reused.
    [042] In the case of treatment with diluted sulfuric acid, the concentration of sulfuric acid is preferably 0.1 to 15% by weight,
    Petition 870190015245, of 02/14/2019, p. 10/24
    11/85 more preferably 0.5 to 5% by weight. The reaction temperature can be adjusted in the range of 100 to 300 ° C, and is preferably adjusted in the range of 120 to 250 ° C. The reaction time can be adjusted in the range of 1 second to 60 minutes. The number of times of treatment is not restricted, and the treatment can be performed 1 or more times. In particular, in cases where the treatment is carried out 2 or more times, the conditions for the first treatment may be different from the conditions for the second and subsequent treatment. As the hydrolyzate obtained by treatment with diluted sulfuric acid contains acid, neutralization is necessary to carry out the hydrolysis reaction with cellulase or to use the hydrolyzate as a raw material for fermentation.
    [043] In the case of hydrothermal treatment, water is added so that the biomass comprising cellulose is contained in 0.1 to 50% by weight, and the treatment is then carried out at a temperature of 100 to 400 ° C for 1 second to 60 minutes. Carrying out the treatment under these temperature conditions, cellulose hydrolysis occurs. The number of times of treatment is not restricted, and the treatment can be performed 1 or more times. In particular, in cases where the treatment is carried out 2 or more times, the conditions for the first treatment may be different from the conditions for the second and subsequent treatment.
    [044] The cellulase used in the present invention is cellulase derived from filamentous fungus. Examples of the filamentous fungus include microorganisms such as Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humicola, Acremonium, Irpex, Mucor and Talaromyces. As these microorganisms secrete cellulase into the culture medium, the culture medium can be used in the same way as cellulase derived from unpurified filamentous fungus, or the culture medium can be purified and formulated to be used as a mixture containing cellulase. derived from filamentous fungus. In cases where cellulase derived
    Petition 870190015245, of 02/14/2019, p. 10/25
    12/85 of the filamentous fungus is used as a purified and formulated product, a substance (s) other than the enzyme, such as a protease inhibitor, dispersant, solubilizer and / or stabilizer can be added to prepare the cellulase formulation .
    [045] The cellulase derived from the filamentous fungus used in the present invention is preferably the cellulase produced by Trichoderma (hereinafter called cellulase derived from Trichoderma). In the present invention, cellulase derived from Trichoderma is preferably cellulase derived from Trichoderma reesei, and specific examples of the preferred microorganisms from Trichoderma from which cellulase is derived include Trichoderma reesei QM9414, Trichoderma reesei QM9123, Trichoderma reesei RichC-30, PC3-7, Trichoderma reesei CL-847, Trichoderma reesei MCG77, Trichoderma reesei MCG80 and Trichoderma viride QM9123 (Trichoderma viride QM9123). Cellulase can also be derived from a mutant strain originating from the microorganism Trichoderma described above, which mutant strain was prepared by mutagenesis using a mutagen, UV irradiation or other similar methods to increase cellulase productivity.
    [046] Filamentous fungus-derived cellulase is an enzyme composition that comprises a plurality of enzyme components such as cellobiohydrolase, endoglucanase, exoglucanase, βglucosidase, xylanase and xylosidase, and this enzymatic composition has an activity to hydrolyze and saccharify cellulose. As cellulase derived from filamentous fungus comprises such a plurality of enzyme components and allows, in cellulose degradation, efficient hydrolysis of cellulose due to its combined effect or complementary effect, preferably cellulase derived from filamentous fungus is used in the present invention.
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    13/85 [047] Cellobiohydrolase is a generic term for cellulases that hydrolyse cellulose in the terminal portions. The group of enzymes belonging to cellobiohydrolase is described as the EC number: EC 3.2.1.91.
    [048] Endoglucanase is a generic term for cellulases that hydrolyze cellulose molecular chains in their central portions. The group of enzymes belonging to the endoglucanase is described as the EC number: EC 3.2.1.4.
    [049] Exoglucanase is a generic term for cellulases that hydrolyze cellulose molecular chains from their terminal portions. The group of enzymes belonging to the exoglucanase is described as the EC number: EC 3.2.1.74.
    [050] β-glucosidase is a generic term for cellulases that act on cell oligosaccharides or cellobiosis. The group of enzymes belonging to β-glucosidase is described as the EC number: EC 3.2.1.21.
    [051] Xylanase is a generic term for cellulases that act on hemicellulose or especially xylan. The group of enzymes belonging to xylanase is described as the EC number: EC 3.2.1.8.
    [052] Xylosidase is a generic term for cellulases that act on xylooligosaccharides. The group of enzymes belonging to xylosidase is described as the EC number: EC 3.2.1.37.
    [053] The components of the cellulase derived from filamentous fungus can be separated using a known method, such as gel filtration, ion exchange or two-dimensional electrophoresis, and the separated components can be subjected to amino acid sequence analysis (Nterminal analysis, analysis C-terminal or mass spectrometry), succeeded by comparing the sequences with a database.
    [054] The enzymatic activity of cellulase derived from filamentous fungus can be evaluated based on its hydrolytic activities in
    Petition 870190015245, of 02/14/2019, p. 10/277
    14/85 polysaccharides, such as Avicel's degradation activity, carboxymethylcellulose (CMC) degradation activity, cellobiose degradation activity, xylan degradation activity and mannan degradation activity. The main components of cellulase involved in Avicel's degradation activity are cellobiohydrolase and exoglucanase, which degrade cellulose in its terminal portions. The main components of cellulase involved in xylan degradation activity are xylanase and xylosidase. The main cellulase component involved in cellobiose degradation activity is β-glucosidase. The main components of cellulase involved in CMC degradation activity are cellobiohydrolase, exoglucanase and endoglucanase. The term "principal" is used here to express that the component (s) is / are involved in degradation to the maximum extent (s) although other enzymatic components are also involved in the degradation.
    [055] As cellulase derived from filamentous fungus, a crude enzyme product is preferably used. The crude enzyme product is derived from the culture supernatant obtained after cultivating a microorganism belonging to a genus of filamentous fungus for an arbitrary period of time in a medium prepared so that the microorganism produces cellulase. The components of the medium to be used are not restricted, and a medium supplemented with cellulose to promote cellulase production can be used in general. As a product of the crude enzyme, the culture liquid can be used as it is, or preferably the culture supernatant processed only with the removal of the filamentous fungus can be used.
    [056] The weight ratios of the enzyme components in the crude enzyme product are not restricted, and, for example, the liquid from the culture derived from Trichoderma reesei contains 50 to 95% by weight of cellobiohydrolase, and
    Petition 870190015245, of 02/14/2019, p. 10/28
    15/85 also contains endoglucanase, β-glucosidase and the like. The microorganisms belonging to Trichoderma produce strong components of cellulase in the culture liquid, while the activity of β-glucosidase in the culture liquid is low, since β-glucosidase is retained in cells or cell surfaces. Therefore, β-glucosidase from a different species or from the same species can be added to the crude enzyme product. As well as β-glucosidase of a different species, β-glucosidase derived from Aspergillus can preferably be used. Examples of Aspergillus-derived β-glucosidase include Novozyme 188, which is commercially available from Novozyme. The method of adding βglucosidase from a different species or from the same species as the crude enzyme product can be a method in which a gene is introduced into a microorganism belonging to Trichoderma to carry out the genetic recombination of the microorganism so that β-glucosidase is produced in the culture liquid, and the microorganism belonging to the Trichoderma is then cultivated, followed by the isolation of the culture liquid.
    [057] In the present invention, the hydrolysis of cellulose with cellulase derived from filamentous fungus is carried out in two stages, that is, primary hydrolysis and secondary hydrolysis. The steps are described below in order.
    [058] The primary hydrolysis in the present invention means that carbohydrase is added to cellulose that has not been brought into contact with carbohydrase, to carry out hydrolysis. The enzyme used for primary hydrolysis can be either the fresh enzyme mentioned later or the recovered enzyme, and in view of the reduction in the amount of enzyme used, especially in the amount of fresh enzyme used, it is preferable to use a mixture of the recovered enzyme and the fresh enzyme.
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    16/85 [059] The reaction temperature during primary hydrolysis is preferably in the range of 40 to 60 ° C, and, especially in cases where Trichoderma-derived cellulase is used, the reaction temperature is most preferably in the range of 45 to 55 ° C.
    [060] The reaction time of primary hydrolysis is preferably in the range of 2 hours to 200 hours. In cases where the reaction time is less than 2 hours, the sugar yield is insufficient, which is not preferred. On the other hand, in cases where the reaction time is greater than 200 hours, the enzyme activity decreases, which is not preferable since, in the secondary hydrolysis mentioned later, the sugar yield is insufficient and the enzyme cannot be recovered .
    [061] The pH during primary hydrolysis is preferably in the range of 4.0 to 5.5. In cases where cellulase derived from Trichoderma is used as cellulase derived from filamentous fungus, the optimum pH of the reaction is 5.0, but, especially in the case of primary hydrolysis, the pH changes during hydrolysis. Therefore, it is preferable to carry out the hydrolysis while maintaining a constant pH using an acid or alkali.
    [062] The primary hydrolyzate contains a liquid of primary sugar and solids, and the solids contain polysaccharide components, such as hemicellulose and undegraded cellulose, and components that cannot be originally degraded with carbohydrase, such as lignin. In addition, a relatively large amount of cellulase derived from filamentous fungus is adhered to the solids. Therefore, in the present invention, to carry out the secondary hydrolysis mentioned later using the polysaccharide components and the cellulase derived from the filamentous fungus contained in the solids obtained by the primary hydrolyzate, the obtained solids are recovered by solid-liquid separation. Examples of the solid-liquid separation method include centrifugation and filtration using a filter press, and, in the
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    17/85 of the present invention, recovery of solids by filtration using a filter press is preferred.
    [063] The reason why filtration using filter press is preferable for solid-liquid separation is that 1) high yield of the sugar liquid can be achieved. The present invention aims to improve sugar recovery and enzyme recovery over conventional techniques. Therefore, the solid-liquid separation method is preferably a method with which larger amounts of liquid sugar components can be recovered at once. The recovery of the components of the sugar liquid through solid-liquid separation can be improved especially by increasing the amount of water that will be added after secondary hydrolysis. However, an increase in the amount of water that will be added generates a reduction in the sugar concentration in the secondary sugar liquid, which is not preferred. Therefore, in view of the suppression of the amount of water used, while achieving a high sugar recovery, the solid-liquid separation is preferably carried out through filtration using a filter press. Another reason why filtration using filter press is preferred is that 2) a transparent filtrate can be obtained. In the present invention, the primary sugar liquid and / or secondary sugar liquid obtained by the solid-liquid separation is / are filtered through an ultrafiltration membrane to recover the enzyme components. The sugar liquid that will be passed through the ultrafiltration membrane preferably contains only small amounts of solids and particulate components in view of preventing the encrustation of the membrane, and in the case of filtration using a filter press, the filtrate contains only small amounts of solids and particulate components and can therefore preferably be used in the present invention.
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    18/85 [064] The secondary hydrolysis in the present invention means that the second hydrolysis is carried out for the solids obtained by the solid-liquid separation of the primary hydrolyzate, using only the cellulase derived from filamentous fungus adsorbed on the solids. That is, in secondary hydrolysis, the hydrolysis of the solids is carried out only with the adsorbed enzyme, without additional addition of carbohydrase.
    [065] Conversely to conventional techniques (where only primary hydrolysis is carried out), the present invention is characterized by the fact that secondary hydrolysis is carried out without additional addition of enzyme, to improve the sugar yield and / or the recovery rate of the enzyme. Sugar production and / or enzyme recovery can also be carried out, of course, in conventional techniques, but by carrying out the secondary hydrolysis of the present invention, it is possible to recover more sugar and enzyme. A main reason for this is to prevent inhibition of the enzyme by removing the sugar produced. The hydrolyzate after primary hydrolysis contains a large amount of sugar components. By performing the solid-liquid separation to remove the sugars (glucose, xylose and oligosaccharides) produced by hydrolysis and subsequently adding water, the concentration of the produced sugars contained in the solution components can be reduced. In this way, inhibition of the enzyme by the products can be avoided, and secondary hydrolysis can be performed sufficiently only with the enzyme adsorbed on the solids. Therefore, even with an equal amount of enzyme as that used in a conventional technique, it is possible to recover more sugar and / or enzyme by performing the secondary hydrolysis of the present invention.
    [066] The amount of water that will be added in the present invention is not restricted, and the addition is preferably carried out so that the concentration of solid before secondary hydrolysis is between 1% in
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    19/85 weight and 20% by weight. In cases where the solid concentration is greater than 20% by weight, and in cases where the solid concentration is less than 1% by weight, the sugar yield and / or the amount of enzyme recovered may be low, the which is inefficient and not preferential.
    [067] The reaction temperature during secondary hydrolysis is preferably in the range 40 to 60 ° C, and, especially in cases where cellulose derived from Trichoderma is used, the reaction temperature is most preferably in the range 40 to 55 ° C, even more preferably 50 ° C.
    [068] The reaction time of the secondary hydrolysis is preferably in the range of 5 to 180 minutes. In cases where the reaction time is less than 5 minutes, the recovery efficiency of the adsorbed enzyme is low, whereas, even in cases where the reaction is carried out for not less than 180 minutes, the recovery efficiency of the adsorbed enzyme does not increase, which is inefficient.
    [069] The pH during secondary hydrolysis is preferably in the range of 6.0 to 8.0. In cases where cellulase derived from Trichoderma is used as cellulase derived from filamentous fungus, the optimal pH of the reaction is 5.0, and, especially in the case of primary hydrolysis, the reaction is preferably carried out at a pH of 5.0. On the other hand, in secondary hydrolysis, as the main objective is the recovery of the adsorbed enzyme, the reaction is preferably carried out at a pH in the range of 6.0 to 8.0, where the recovery efficiency of the adsorbed enzyme is high. At a pH less than 6.0, the degree of recovery of the enzyme decreases, while with a pH above 8.0, carbohydrase is deactivated, which is not preferred. That is, at a pH in the range of 6.0 to 8.0, the degree of carbohydrate deactivation is extremely low and the efficiency of carbohydrate recovery can be high.
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    20/85 [070] The secondary hydrolyzate contains a secondary sugar liquid and solids, and, similarly to the case of primary hydrolysis, these components can be separated from each other by solid-liquid separation, preferably filtration using a filter press.
    [071] In secondary hydrolysis, one or more compounds selected from non-ionic surfactants, amino acids, inorganic salts (except calcium salts) and hydrophilic organic solvents can be added. Adding these compound (s), one or more of the sugar yield, the amount of the enzyme recovered and the activity of the recovered enzyme can be increased. In particular, in cases where the activity of the recovered enzyme is high, the amount of fresh enzyme that will be added by reusing the recovered enzyme can be reduced, which is economically preferable.
    [072] Secondary hydrolysis can be carried out in the presence of a surfactant, and the surfactant is preferably a non-ionic surfactant, because, in cases where a cationic surfactant, anionic surfactant or amphoteric surfactant is used, the surfactant promotes the deactivation of carbohydrase and has an inhibitory action on the secondary hydrolysis reaction. In addition, the activity of the recovered enzyme is also reduced, which is not preferred. On the other hand, with a nonionic surfactant, a high efficiency of sugar yield and a high recovery efficiency of the enzyme can be obtained, therefore, a nonionic surfactant is preferably used.
    [073] The non-ionic surfactant is also called non-ionic surfactant, and is a surfactant whose hydrophilic fraction consists of a non-electrolyte. Specific examples of the nonionic surfactant include polyoxyethylene alkyl ethers, polyoxypropylene block copolymers, polyoxyethylene alkyl alkyl ethers, polyoxyethylene fatty acid esters, fatty acid esters
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    21/85 sorbitan, polyoxyethylene fatty acid esters of sorbitan, nonyl phenyl ethers of polyoxyethylene, naphthyl ethers of polyoxyethylene, octylphenyl ethers of polyoxyethylene, polyoxyethylene alkyl amines, fatty acid esters of glycerin and polyoxyethylene oxides of the series of acetylene and ethylene oxides from the series can be used individually or as a mixture of two or more of those mentioned. The nonionic surfactant is preferably a polyoxypropylene block copolymer. The molecular weight of the polyoxypropylene block copolymer is preferably in the range of 500 to 15000.
    [074] The nonionic surfactant is preferably added at a concentration in the range of 0.05 to 5% by weight. In cases where the concentration is less than 0.05% by weight, the efficiency of recovery of carbohydrase is low, while in cases where the concentration is above 5% by weight, the deactivation of carbohydrase is encouraged, which it is not economically advantageous and is therefore not preferential.
    [075] Secondary hydrolysis can be carried out in the presence of an inorganic salt (s), and examples of inorganic salt (s) that can be used include sodium salts, potassium salts, salts magnesium, sulfuric acid salts, ammonium salts, hydrochloric acid salts, phosphoric acid salts, acetic acid salts and nitric acid salts. Examples of more preferred inorganic salts include sodium chloride, sodium acetate, sodium sulfate, sodium hydrogen sulfate, sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium chloride, dipotassium hydrogen phosphate, ammonium sulfate, magnesium chloride and magnesium sulfate. Among these, sodium chloride, sodium sulfate and sodium hydrogensulfate, which are sodium salts; and magnesium chloride and magnesium sulfate, which are magnesium salts; are most preferred. By adding an inorganic salt (s) of it, the degradation activity of Avicel and the degradation activity of xylan in the recovered enzyme can be increased.
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    22/85 [076] Additionally, as an alternative to these inorganic salts, sea water can be used. Seawater is an aqueous solution of inorganic salt containing 2.6 to 2.7% sodium chloride, 0.3 to 0.4% magnesium chloride, 0.1 to 0.2% sulfate magnesium and about 0.07% potassium chloride and that occurs in nature in greater quantity. Therefore, sea water can be used as an aqueous solution of inorganic salt in secondary hydrolysis. The pH of seawater is largely dependent on its salt composition, and is generally in the range of 8.2 to 8.5. Sea water can be used for secondary hydrolysis without changing the pH or after adjusting the pH to an arbitrary value. It is preferable to adjust the pH to a value in the range of 5 to 8.3 in view of the increased cellulase activity of the recovered enzyme. For pH adjustment, a common acid, such as sulfuric acid or hydrochloric acid, can be used, and the acid is not restricted.
    [077] Additionally, as an alternative to these (in) salt (s) inorganic (s), the ash prepared by submitting the biomass containing cellulose, a pretreated biomass product containing cellulose, the residue of saccharification can be used. obtained after the hydrolysis of biomass containing cellulose, or others similar, to the combustion of the heater. These ashes contain a large amount of potassium salts, and an aqueous solution of inorganic salt can be prepared by dissolving the salts in water.
    [078] The inorganic salt (s) is / are preferably added in a concentration in the range of 0.05 to 5% by weight. In cases where the concentration is less than 0.05% by weight, the recovery efficiency of carbohydrase is low, while in cases where the concentration is above 5% by weight, the deactivation of carbohydrase is stimulated, which does not it is economically advantageous, therefore, not preferential. In cases where sea water is used as the aqueous salt solution
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    23/85 inorganic, the sea water dilution rate is preferably adjusted in the range of 1/10 to 1.
    [079] Secondary hydrolysis can be carried out in the presence of a hydrophilic organic solvent (s). The hydrophilic organic solvent in the present invention means one that exhibits a solubility of not less than 100 g / L in water at 20 ° C. On the other hand, an organic solvent that exhibits a solubility of less than 100 g / L under the above conditions is called a hydrophobic organic solvent. Examples of hydrophobic organic solvents include, but are not limited to, 1-butanol (74 g / L), 1-pentanol (27 g / L), 1-hexanol (5.8 g / L), ethyl acetate (83 g / L), hexane (trace value) and chloroform (trace value). Representative examples of the hydrophilic organic solvent in the present invention include methanol, ethanol, 1-propanol, isopropanol, dimethylsulfoxide, N, N-dimethylformamide, acetone, acetonitrile, ethylene glycol and glycerin. By adding a hydrophilic organic solvent, Avicel's degradation activity of the recovered enzyme can be enhanced, which is preferable.
    [080] The hydrophilic organic solvent (s) mentioned is / are preferably added in a concentration in the range of 0.05 to 5% by weight. In cases where the concentration is less than 0.05% by weight, the recovery efficiency of carbohydrase is low, while in cases where the concentration is above 5% by weight, the deactivation of carbohydrase is stimulated, which does not it is economically advantageous, therefore, not preferential.
    [081] Secondary hydrolysis can be performed in the presence of an amino acid (s), and examples of the amino acid (s) that can be used include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, acid glutamic, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine, and their derivatives. Among these amino acids, alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine,
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    24/85 methionine, phenylalanine, proline, serine, threonine, tryptophan and valine, which have high solubility in water, are preferred. Arginine, cysteine, glutamic acid, histidine and lysine, with which it is possible to obtain the recovered enzyme with high Avicel degradation activity, are of utmost preference.
    [082] The amino acid (s) cited is / are preferably added in a concentration in the range of 0.05 to 5% by weight. In cases where the concentration is less than 0.05 by weight, the recovery efficiency of carbohydrase is low, while in cases where the concentration is above 5% by weight, the deactivation of carbohydrase is stimulated, which is not economically advantageous, therefore, not preferential.
    [083] In the present invention, the primary sugar liquid and / or secondary sugar liquid is / are filtered through an ultrafiltration membrane, and the carbohydrase is separated / recovered from the feed side, and a sugar solution is recovered from the permeate side. The cut molecular weight of the ultrafiltration membrane used in the present invention is not restricted, as long as it allows the permeation of glucose (molecular weight, 180), which is a monosaccharide, and allows the enzyme to be blocked. More specifically, the cut molecular weight can be in the range of 500 to 50000, and the ultrafiltration membrane has a cut molecular weight preferably in the range of 5000 to 50000, more preferably in the range of 10,000 to 30000. Examples of the material that can be used for the functional membrane of the ultrafiltration membrane include polyether sulfone (PES), polysulfone (PS), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), regenerated cellulose, cellulose, cellulose ester, sulfonated polysulfone, sulfonated polyether sulfone, polyolefin, polyvinyl alcohol, polymethyl methacrylate and polytetrafluoroethylene. As regenerated cellulose, cellulose and cellulose ester are subjected to degradation by cellulase, an ultrafiltration membrane that uses a material
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    25/85 synthetic polymer, such as PES or PVDF, is preferably used. Examples of the method of filtration through an ultrafiltration membrane include "dead end" filtration and cross-flow filtration, and the preferred method is cross-flow filtration in order to eliminate scale encrustation. Examples of the shape of the ultrafiltration membrane that can be used appropriately include smooth membrane, spiral membrane, tubular membrane and hollow fiber membrane. Specific examples of the ultrafiltration membrane include Type G-5, Type G-10, Type G-20, Type G50, Type PW and Type HWS UF, manufactured by DESAL; HFM-180, HFM-183, HFM-251, HFM-300, HFM-116, HFM-183, HFM-300, HFK-131, HFK-328, MPTU20, MPS-U20P and MPS-U20S, manufactured by KOCH; SPE1, SPE3, SPE5, SPE10, SPE30, SPV5, SPV50 and SOW30, manufactured by Synder; UF series Microza (trademark) products, manufactured by Asahi Kasei Corporation, which have cut molecular weights from 3000 to 100000; and NTR7410 and NTR7450, manufactured by Nitto Denko Corporation.
    [084] In cases where a compound (s), such as a non-ionic surfactant (s), inorganic salt (s), hydrophilic organic solvent (s) (s), amino acid (s) and / or the like is / are added for secondary hydrolysis, the secondary sugar liquid naturally contains these added compounds. These compounds may have inhibitory actions on the subsequent fermentation stage, depending on their types and the quantities added. In such a case, only the recovered enzyme can be separated / recovered from the secondary sugar liquid using an ultrafiltration membrane and the sugar liquid containing inorganic salts obtained from the permeate side can be treated as a waste liquid.
    [085] In the present invention, it is preferable to filter the primary sugar liquid and the secondary sugar liquid through an ultrafiltration membrane and additionally filter the sugar liquid obtained from the side
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    26/85 permeated through a reverse osmosis membrane and / or nanofiltration membrane. In the present invention, the secondary sugar liquid is likely to have a lower sugar concentration than the primary sugar liquid because, for example, 1) how the secondary sugar liquid is produced through the hydrolysis reaction using only the carbohydrate adhered to the solids , the absolute amount of carbohydrase is less; and 2) the efficiency of hydrolysis of lignocellulose that remained as solids is low. Therefore, in cases where only the secondary sugar liquid, or a mixture of the secondary sugar liquid and the primary sugar liquid is used in the subsequent fermentation step, the concentration of the fermentation product may be low due to the low sugar concentration . However, by filtering the sugar liquid through a reverse osmosis membrane and / or nanofiltration membrane, the reduction in the sugar concentration in the sugar liquid can be avoided. The sugar concentration, in the present case, means the total amount of monosaccharide components, especially glucose and xylose. The concentration rate at that sugar concentration is not restricted, as long as the concentration is carried out to achieve an appropriate concentration for the subsequent fermentation step. The concentration of sugar in the sugar solution before the concentration is not restricted, and is preferably in the range of 10 g / L to 100 g / L. The sugar concentration after the concentration is not restricted, and in general the sugar liquid can be preferably used in the subsequent fermentation step in cases where the sugar concentration is from 50 g / L to 200 g / L.
    [086] Examples of the nanofiltration membrane or reverse osmosis membrane material that can be used in the present invention include polymeric materials, such as cellulose acetate polymers, polyamides, polyesters, polyimides, vinyl polymers and polysulfones. The membrane is not restricted to a membrane consisting of only one of the
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    27/85 materials, and can be a membrane comprising a plurality of materials for the membrane.
    [087] As a nanofiltration membrane to be used in the present invention, a spiral membrane element is preferred. Specific examples of the preferred nanofiltration membrane element include a GE Sepa cellulose acetate nanofiltration membrane element, manufactured by GE Osmonics; NF99 and NF99HF nanofiltration membrane elements, manufactured by Alfa-Laval, which have functional polyamide layers; nanofiltration membrane elements NF-45, NF-90, NF-200, NF-270 and NF-400, manufactured by FilmTec Corporation, which have functional layers of cross-linked piperazine polyamide; and SU-210, SU-220, SU-600 and SU-610 nanofiltration membrane elements, manufactured by Toray Industries, Inc., which comprise a UTC60 nanofiltration membrane, manufactured by the same manufacturer, which comprises a cross-linked piperazine polyamide as the main component. A nanofiltration membrane element is most preferably NF99 or NF99HF; NF-45, NF-90, NF-200 or NF-400; or SU-210, SU-220, SU-600 or SU-610. A nanofiltration membrane element is even more preferably SU-210, SU-220, SU-600 or SU-610.
    [088] As a reverse osmosis membrane to be used in the present invention, a spiral membrane element is preferred as in the case of the nanofiltration membrane. Specific examples of the preferred reverse osmosis membrane element include polyamide reverse osmosis membrane modules manufactured by TORAY INDUSTRIES, INC. SU710, SU-720, SU-720F, SU-710L, SU-720L, SU-720LF, SU-720R, SU-710P and SU-720P, which are low-pressure modules, as well as SU-810, SU-820 , SU820L and SU-820FA that contain UTC70 as a reverse osmosis membrane, which are high pressure modules; reverse osmosis membranes
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    28/85 cellulose acetate manufactured by the same manufacturer SC-L100R, SC-L200R, SC-1100, SC-1200, SC-2100, SC-2200, SC-3100, SC-3200, SC-8100 and SC8200; NTR-759HR, NTR-729HF, NTR-70SWC, ES10-D, ES20-D, ES20-U, ES15-D, ES15-U and LF10-D, manufactured by Nitto Denko Corporation; RO98pHt, RO99, HR98PP and CE4040C-30D, manufactured by Alfa-Laval; GE Sepa, manufactured by GE; and BW30-4040, TW30-4040, XLE-4040, LP-4040, LE-4040, SW30-4040 and SW30HRLE-4040, manufactured by FilmTec Corporation.
    [089] The apparatus for carrying out the method of the present invention explained above for producing a sugar liquid is described below. The apparatus for carrying out the method for producing a sugar liquid of the present invention must comprise as constituents at least: a stirring tank (2) to carry out primary hydrolysis; secondary hydrolysis tank (28) or filtration device using filter press (8) to perform secondary hydrolysis; solid-liquid separation device (s) (25, 30) for the primary hydrolyzate and for the secondary hydrolyzate; and an ultrafiltration membrane device (12, 33) for separating the carbohydrase and the sugar liquid from the primary sugar liquid and / or the secondary sugar liquid. To describe the modalities of this type of device, specific examples are shown in Figure 2 to Figure 8 and in Figure 10 to Figure 16. The devices in Figure 2 to Figure 8 and Figure 10 to Figure 16 were classified in Configuration 1 to Configuration 4 with based on their characteristics. Configuration 1 is a device configuration in which secondary hydrolysis is carried out in a filtration tank using a filter press (8), and corresponds to Figure 2 to Figure 8.
    [090] Configuration 1 is a mode in which water is circulated to the filtration chamber using a filter press, and is an apparatus configuration that allows the secondary hydrolysis of the present invention, provided that the apparatus has a filtration device using filter press 8
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    29/85 for solid-liquid separation. This configuration has the advantage of the simplified constitution of the device and the cost with the device, therefore, it can be eliminated. However, it has the disadvantage that the primary sugar liquid and the secondary sugar liquid are contaminated within the device.
    [091] Configuration 2 is an apparatus configuration comprising a secondary hydrolysis tank (28) for performing secondary hydrolysis. Configuration 2 comprises a stirring tank (2) and a secondary hydrolysis tank (28) separately. Configuration 2 has the advantage that the solids can be resuspended in the secondary hydrolysis tank (28) and the efficiency of the secondary hydrolysis is high. Depending especially on the types and concentrations of compounds added for secondary hydrolysis, the compounds may exhibit inhibitory actions on the subsequent fermentation of a sugar liquid. Therefore, because it avoids contamination of the primary sugar liquid and the secondary sugar liquid together, this configuration is advantageous because it has, as in Configuration 2, the secondary hydrolysis tank (28) dedicated to the secondary hydrolysis separately from the tank stirring (2) to carry out primary hydrolysis, and also having a solid-liquid separation (30) dedicated to the secondary sugar liquid and an ultrafiltration membrane device (33) dedicated to the secondary sugar liquid. However, Configuration 2 has a disadvantage, as the total number of equipment, including the secondary hydrolysis tank (28), increases, the cost of the equipment also increases.
    [092] Configuration 3, which is a device configuration in which secondary hydrolysis is carried out in the stirring tank (2) where primary hydrolysis is also carried out, is shown in a figure. Configuration 3 is a device configuration in which the hydrolyzate obtained in the
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    30/85 agitation tank (2) is subjected to solid-liquid separation and then returned to the agitation tank (2), followed by the addition of water to the secondary hydrolysis. Configuration (3) has the advantage that the number of equipment can be the lowest and the cost of the equipment can be reduced. However, Configuration 3 has the disadvantage of contaminating the primary sugar liquid and the secondary sugar liquid within the apparatus.
    [093] The Examples of Configuration 4, which is a modality in which a microfiltration membrane device 36 is positioned between a solid-liquid separation device (25) and an ultrafiltration membrane device (12) are partially shown in the figures . The placement of the microfiltration membrane device has the advantage of removing insoluble microparticles, which could not be removed sufficiently by the solid-liquid separation, and the obstruction of the membrane in the ultrafiltration membrane device (12) can be reduced in one step later.
    Table 1
    Configuration Device features Corresponding figures 1 Device configuration where secondary hydrolysis is performed on a filtration tank device using filter press Figure 2 to Figure 8 2 Device configuration where secondary hydrolysis is performed in a secondary hydrolysis tank Figure 10 to Figure12 3 Device configuration in which secondary hydrolysis is carried out in a stirring tank that is also used for primary hydrolysis Figure 13 4 Device configuration in which a microfiltration membrane device is placed upstream of an ultrafiltration membrane device (partial diagram) Figure 14 to Figure16
    [094] Configuration 1, which is a modality in which secondary hydrolysis is performed on a filtration device using a filter
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    31/85 press, is described below using the schematic diagrams shown in Figure 2 to Figure 8.
    [095] Examples of the apparatus for carrying out the method for producing a sugar liquid of the present invention include an apparatus comprising as a constituent a stirring tank for primary hydrolysis (2), filtration device using a filter press (8) which has a warm water supply tank (6), circulation line (10) to circulate the filtrate from the filtration device using filter press (8) to the warm water supply tank (6), and ultrafiltration membrane device ( 12) to separate the carbohydrase and the sugar solution from the primary sugar liquid and / or secondary sugar liquid. The apparatus for the method for producing a sugar liquid of the present invention is described below with reference to the examples of the apparatus shown in the figures.
    [096] Figure 2 and Figure 3 are schematic diagrams showing the devices, each using a filtration device using a filter press (8) that has a warm water inlet (15) and a hydrolyzate inlet (14) shown in Figure 4 separately. Figure 5 and Figure 6 are schematic diagrams showing the devices, each using a filtration device using a filter press (8) that has a warm water inlet together with a hydrolyzate inlet (21) shown in Figure 7. Figure 2 and Figure 5 are schematic diagrams showing the devices, each of which has a stirring tank (2) and a warm water supply tank 6 separately. On the other hand, Figure 3 and Figure 6 are schematic diagrams showing the devices, each of which uses a stirring tank (2) also as a warm water supply tank.
    [097] The device shown in Figure 2 is described below in detail. The stirring tank (2) to perform the primary hydrolysis has a
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    32/85 inlet (3) for supplying cellulose, the stirring device (4) for stirring / mixing lignocellulose, and the thermostat (1) for maintaining the temperature of the stirring tank. The primary hydrolyzate obtained in the stirring tank (2) is fed into the filtration device using a filter press (8) through a hydrolyzate inlet (14). In the filtration device using a filter press (8), the solid-liquid separation is carried out by compression with a compressor (9), and the warm water is supplied by a warm water supply tank (6) to the solids retained in the chamber. filtration using filter press through a warm water inlet (15). The warm water supply tank (6) has a water supply line (5), the warm water supply tank thermostat (7) to maintain the warm water temperature at a predetermined value, and a circulation line (10) to circulate the filtrate obtained by filtration using a filter press. The primary sugar liquid and / or the secondary sugar liquid obtained by filtration using a filter press is retained in a filtrate recovery tank (11), and filtered through an ultrafiltration membrane device (12). The recovered carbohydrase is recovered and / or reused through a carbohydrase recovery line.
    [098] The device shown in Figure 3 is described below in detail. The stirring tank (2) for performing primary hydrolysis has an inlet (3) for cellulose feeding, the stirring device (4) for stirring / mixing the cellulose, and a thermostat (1) for maintaining the temperature of the agitation. The primary hydrolyzate obtained in the stirring tank (2) is fed into the filtration device using filter press 8 through a hydrolyzate inlet (14). In the filtration device using a filter press (8), the solid-liquid separation is carried out by compression with a compressor (9), and warm water is supplied by a warm water supply tank (6) to the solids retained in the filtration chamber. using filter press through a
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    33/85 warm water inlet (15). A hydrolyzate inlet (14) and a warm water inlet (15) are connected to the filtration device using a filter press (8), and the flow can be bypassed with a valve. The primary sugar liquid and / or the secondary sugar liquid obtained by filtration using a filter press is retained in a filtrate recovery tank (11), and filtered through an ultrafiltration membrane device (12). The recovered carbohydrase is recovered and / or reused through a carbohydrase recovery line.
    [099] The device shown in Figure 5 is described below in detail. The stirring tank (2) for performing primary hydrolysis has an inlet (3) for cellulose supply, the stirring device (4) for stirring / mixing cellulose, and a thermostat (1) for maintaining the temperature of the stirring tank. The primary hydrolyzate obtained in the stirring tank (2) is fed into the filtration device using a filter press (8) through a warm water inlet together with a hydrolyzate inlet (21). In the filtration device using a filter press (8), the solid-liquid separation is carried out by compression with a compressor (9), and warm water is supplied by a warm water supply tank (6) to the solids retained in the filtration chamber. using a filter press through the entry of warm water in conjunction with the entry of hydrolyzate (21). The warm water supply tank (6) has a water supply line (5), the warm water supply tank thermostat (7) to maintain the warm water temperature at a predetermined value, and a circulation line (10) to circulate the filtrate obtained by filtration using a filter press. The primary sugar liquid and / or the secondary sugar liquid obtained by filtration using a filter press is / are retained in a filtrate recovery tank (11), and filtered through an ultrafiltration membrane device (12). The recovered carbohydrase is
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    34/85 recovered and / or reused through a carbohydrase recovery line.
    [0100] The device shown in Figure 6 is described below in detail. The stirring tank (2) for carrying out primary hydrolysis has an inlet (3) for feeding cellulose, the stirring device (4) for stirring / mixing cellulose, and a thermostat (1) for maintaining the temperature of the stirring tank. . The primary hydrolyzate obtained in the stirring tank (2) is fed into the filtration device using a filter press (8) through a warm water inlet together with a hydrolyzate inlet (21). In the filtration device using the filter press (8), solid-liquid separation is performed by compression with a compressor (9), and warm water is supplied by the stirring tank (2 to the solids retained in the filtration chamber using the filter press through the warm water inlet together with hydrolyzate inlet (21). The primary sugar liquid and / or the secondary sugar liquid obtained by filtration using a filter press is / are retained in a filtrate recovery tank (11), and filtered through an ultrafiltration membrane device (12) .The recovered carbohydrase is recovered and / or reused through a carbohydrase recovery line.
    [0101] In the equipment described above, secondary hydrolysis can be carried out by subjecting the primary hydrolyzate to filtration using a filter press and feeding and / or circulating warm water from 40 to 60 ° C in the tank of the filtration chamber that retains the obtained solids. As the solids after filtration using a filter press have a low moisture content and low fluidity, performing secondary hydrolysis in a separate stirring vessel or similar requires power to provide energy for further dispersion of the solids. Providing warm water preheated to a temperature in the range of 40 to 60 ° C in the warm water supply tank 6 to the filtration chamber using a filter press, the activity of the enzyme components adsorbed to the
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    35/85 solids can be increased, so that the secondary hydrolysis of the present invention can be carried out. In cases where the amount of warm water fed is very large, the sugar concentration in the secondary sugar liquid is very low, which is not preferred. On the other hand, in cases where the amount of warm water fed is very small, the reaction temperature in the filtration chamber cannot be sufficiently maintained, which is not preferred. It should be noted that by heating the water once fed to 40 to 60 ° C and circulating the water again, the reaction temperature can be maintained, and the sugar concentration in the secondary sugar liquid can be increased. The time period for feeding and / or circulating warm water is preferably in the range of 5 minutes to 180 minutes. In cases where the time period is below 5 minutes, secondary hydrolysis cannot be sufficiently performed, while in cases where the time period is above 180 minutes, the sugar production rate tends to be saturated, the which is not preferred from an energy point of view.
    [0102] The filtration device using filter press is shown in Figure 4 and Figure 7 as schematic diagrams. In the device shown in Figure 4, the primary hydrolyzate is fed through the hydrolyzate inlet (14) to the filtration chamber using a filter press (20), and the solid-liquid separation is carried out by compression with a pressing plate (19). After that, warm water is fed through the warm water inlet (15) to bring the warm water into contact with the solids (primary hydrolyzate) (18), then it is filtered through a filter cloth (17). The filtrate is additionally circulated through a thermostat, and again fed into the filtration chamber using a filter press through the entry of warm water (15). Enabling this circulation, secondary hydrolysis can be carried out in the filtration chamber using a filter press. Figure 7 is a diagram
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    36/85 schematic showing a method for providing warm water through a warm water inlet in conjunction with hydrolyzate inlet (21). That is, the primary hydrolyzate is fed through the entry of warm water together with the hydrolyzate (21) into the filtration chamber using a filter press (20) and subjected to compression with a pressing plate (19), and with that the solid-liquid separation is performed. After that, the warm water is fed through the warm water inlet together with the hydrolyzate inlet (21) to bring the warm water into contact with the solids (primary hydrolyzate) (18), then being filtered through a cloth. filter (17). The filtrate is additionally circulated through a thermostat, and again fed into the filtration chamber using a filter press through the inlet of warm water together with the inlet of hydrolyzate (21). Enabling this circulation, secondary hydrolysis can be carried out in the filtration chamber using a filter press. The time period for feeding and / or circulating warm water to the filtration chamber using a filter press is preferably in the range of 5 minutes to 180 minutes. In cases where the time period is below 5 minutes, secondary hydrolysis cannot be sufficiently performed, while in cases where the time period is above 180 minutes, the sugar production rate tends to be saturated, the which is not preferred from an energy point of view. In cases where the amount of warm water fed is very large, the sugar concentration in the secondary sugar liquid is very low, which is not preferred. On the other hand, in cases where the amount of warm water fed is very small, the reaction temperature in the filtration chamber cannot be maintained sufficiently, which is not preferred. In such cases, by heating the water once fed to 40 to 60 ° C and circulating the water again, the reaction temperature can be
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    37/85 maintained, and the sugar concentration in the secondary sugar liquid can be increased.
    [0103] Figure 8 is a schematic diagram showing an apparatus in which a sugar concentration device that has a reverse osmosis membrane and / or nanofiltration membrane to concentrate the sugar liquid is additionally attached to the apparatus shown in Figure 2. More specifically, this apparatus comprises, on the filtrate side of the ultrafiltration membrane device (12), a sugar solution tank (22); nanofiltration membrane device and / or reverse osmosis membrane device (23) connected to it by a pump; and a filtrate line (24). In cases where the nanofiltration membrane device and / or the reverse osmosis membrane device is / are connected to the apparatus shown in Figure 3, 5 or 6, the nanofiltration membrane device and / or the osmosis membrane device Reverse can be connected, just like in Figure 8, downstream (downstream) of the ultrafiltration membrane device (12), which is included in the device shown in Figure 3, 5 or 6 similarly to the device shown in Figure 2.
    [0104] Configuration 2, which is a modality in which the secondary hydrolysis is carried out in a secondary hydrolysis tank, is described below with reference from Figure 10 to Figure 12.
    [0105] Figure 10 is a diagram showing an example of an apparatus system that has a stirring tank for primary hydrolysis (2) and a secondary hydrolysis tank (28) separately. The solid-liquid separation device (25) is not restricted, as long as it allows the solid-liquid separation of the primary hydrolyzate using a centrifuge, filter press, belt filter or the like. The solids obtained with the solid-liquid separation device (25) are transferred to the secondary hydrolysis tank (28) by a solid transfer medium (26). The means of
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    38/85 solid transfer (26) is not restricted, as long as it is suited to the properties of the solids, and examples of the medium include a conveyor belt and a screw pump. The secondary hydrolysis tank (28) comprises at least one thermostat (2) (secondary hydrolysis tank) (27) for carrying out the secondary hydrolysis. The secondary hydrolysis tank (28) can further comprise a stirring device (2) (secondary hydrolysis tank) (29) for mixing the solids by stirring. The secondary hydrolysis tank (28) further comprises a solid-liquid separation device (2) (secondary hydrolysis tank) (30) for carrying out the solid-liquid separation of the secondary hydrolyzate. The secondary sugar liquid separated via the solid-liquid separation device (2) (secondary hydrolysis tank) (30) is transferred to a secondary sugar liquid recovery tank (32). The secondary sugar liquid in the secondary sugar liquid recovery tank (32) is filtered through a secondary sugar liquid ultrafiltration membrane device (33) to recover the enzyme.
    [0106] Figure 11 shows an embodiment of an apparatus system that has a stirring tank (2) to perform primary hydrolysis and a secondary hydrolysis tank (28) separately, and a solid-liquid separation device (25) and a solid-liquid separation device (2) (secondary hydrolyzate) (30) separately, while an ultrafiltration membrane device (12) is shared by the primary sugar liquid and the secondary sugar liquid. As in Figure 10, the secondary hydrolyzate obtained in the secondary hydrolysis tank (28) is subjected to solid-liquid separation in the solid-liquid separation device 2 (secondary hydrolyzate) (30), and transferred to a filtrate tank (11 ) via a secondary sugar liquid transfer line (34). The primary sugar liquid and the secondary sugar liquid recovered in the
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    39/85 filtrate tank (11) are filtered through an ultrafiltration membrane (12) immediately or in sequence, and the enzyme and sugar are then separated.
    [0107] Figure 12 shows an embodiment of an apparatus system that has a stirring tank (2) to perform primary hydrolysis and a secondary hydrolysis tank (28) separately, as a solid-liquid separation device (25) and an ultrafiltration membrane device (12) are commonly used by the primary sugar liquid and the secondary sugar liquid. The secondary hydrolyzate obtained in a secondary hydrolysis tank (28) is transferred to a solid-liquid separation device (25) through a secondary hydrolyzate transfer line (35), and separated into secondary and solid sugar liquid. The primary sugar liquid and the secondary sugar liquid separated in the solid-liquid separation device (25) are recovered in a filtrate recovery tank (11) and filtered through an ultrafiltration membrane (12) immediately or in sequence, and the enzyme and sugar are then separated.
    [0108] Configuration 3, which is a modality in which secondary hydrolysis is carried out in a tank that is also used for primary hydrolysis, is described below with reference to Figure 13.
    [0109] The apparatus shown in Figure 13 is intended for a modality in which the primary hydrolyzate obtained in a stirring tank (2) to perform the primary hydrolysis is separated by a solid-liquid separation device (25), and the obtained solids are circulated to the primary hydrolysis tank through a transfer line of the secondary hydrolyzate (35), followed by the execution of the secondary hydrolysis in the primary hydrolysis tank. The primary sugar liquid and the secondary sugar liquid separated in the solid-liquid separation device (25) are
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    40/85 collected in a filtrate recovery tank (11), and filtered through an ultrafiltration membrane device (12) to separate the enzyme and the sugar.
    [0110] The device configurations in which a microfiltration membrane device is placed upstream of an ultrafiltration membrane device are described below with reference to Figure 14 to Figure 16. Figure 14 shows a modality (partial diagram) in which a microfiltration membrane device (36) is placed upstream of an ultrafiltration membrane device (12). The microfiltration membrane device (36) is not restricted, as long as it can remove insoluble microparticle components contained in the primary sugar liquid and / or the secondary sugar liquid obtained in the solid-liquid separation device (33) or in the filtration using filter press (8), and examples of the microfiltration membrane device (36) include microfiltration membranes that have an average pore size in the range of 0.01 pm to 1 pm. The filtration method on the microfiltration membrane device (36) can be cross-flow filtration (Figure 15) or “dead end” filtration (Figure 16).
    [0111] Figure 15 shows an embodiment of the microfiltration membrane device (36) for performing microfiltration through cross-flow filtration. The primary sugar liquid and / or the secondary sugar liquid can be stored in a microfiltration membrane crude liquid tank (37) and filtered through a microfiltration membrane (38) while being circulated by a pump.
    [0112] Figure 16 shows a modality in which microfiltration is performed through “dead end” filtration. The primary sugar liquid and / or the secondary sugar liquid is / are stored in a microfiltration membrane crude liquid tank (37) and filtered through a
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    41/85 microfiltration membrane (38). In the case of “dead end” filtration, a compressed air supply device (39) that performs a bubble wash of the membrane surface can be supplied as required, and an inverted wash pump (40) for inverted washing can be arranged. Inverted washing can be performed with the filtrate recovered in a microfiltrate recovery tank (41), or, in some cases, with a membrane washing liquid or common liquid agent. The microfiltration membrane (38) can be in the form of a smooth membrane or a hollow fiber membrane. The hollow fiber membrane can be an internal pressure membrane or an external pressure membrane.
    Examples [0113] The present invention is more specifically described below by way of Examples. However, the present invention is not restricted to such Examples.
    (Reference Example 1) Preparation of Pretreated Cellulose
    1. Preparation of Pretreated Cellulose 1 (Treatment with Dilute Sulfuric Acid) [0114] Rice straw was used as cellulose. The cellulose was soaked in an aqueous solution of 1% sulfuric acid, and subjected to treatment using an autoclave (manufactured by Nitto Koatsu Co., Ltd.) at 150 ° C for 30 minutes. After the treatment, the solid-liquid separation was carried out to separate the sulfuric acid-treated cellulose from the aqueous sulfuric acid solution (hereinafter referred to as “diluted sulfuric acid treatment liquid”). Subsequently, the cellulose treated with sulfuric acid was mixed with the liquid of the treatment with diluted sulfuric acid by stirring, so that the concentration of the solid content was 10%
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    42/85 by weight, and the pH was adjusted to about 5 with sodium hydroxide. The resulting mixture was used in the Examples below as the pre-treated cellulose 1.
    2. Preparation of Pretreated Cellulose 2 (Treatment with Ammonia) [0115] Rice straw was used as cellulose. The cellulose was fed to a compact reactor (manufactured by Taiatsu Techno Corporation, TVS-N2 30 ml), and cooled with liquid nitrogen. In this reactor, the ammonia gas was drained, and the sample was completely soaked in liquid ammonia. The reactor lid was closed, and the reactor was left at room temperature for about 15 minutes. Subsequently, the reactor was processed in an oil bath at 150 ° C for 1 hour. After that, the reactor was removed from the oil bath, and the ammonia gas was released in a chapel, followed by the internal evacuation of the reactor at 10 Pa with a vacuum pump, thereby drying the cellulose. The resultant was used in the Examples below as the pretreated cellulose 2.
    3. Preparation of Pretreated Cellulose 3 (Hydrothermal Treatment) [0116] Rice straw was used as cellulose. The cellulose was soaked in water, and subjected to treatment using an autoclave (manufactured by Nitto Koatsu Co., Ltd.) at 180 ° C for 20 minutes with stirring. The treatment was carried out at a pressure of 10 MPa. After the treatment, the solid-liquid separation was performed by centrifugation (3000 G) to separate the processed biomass component from the solution component (hereinafter referred to as “hydrothermally treated liquid”). The processed biomass component was used in the Examples below as pretreated cellulose 3.
    (Reference Example 2) Measurement of Sugar Concentration [0117] The concentrations of glucose and xylose contained in the sugar liquid were measured under the HPLC conditions described below based on comparison with standard samples.
    [0118] Column: Luna NH2 (manufactured by Phenomenex, Inc.)
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    43/85 [0119] Mobile phase: MilliQ: acetonitrile = 25:75 (flow rate, 0.6 mL / minute) [0120] Reaction solution: None [0121] Detection method: IR (differential refractive index) [0122] Temperature: 30 ° C (Reference Example 3) Preparation of Trichoderma-derived Cellulase [0123] Trichoderma-derived cellulase was prepared using the following method.
    Pre-culture [0124] A mixture of 5% millhocin (w / vol), 2% glucose (w / vol), 0.37% ammonium tartrate (w / vol), 0.14% ( w / vol) ammonium sulfate, 0.2% (w / vol) potassium dihydrogen phosphate, 0.03% (w / vol) calcium chloride dihydrate, 0.03% (w / vol) sulfate magnesium heptahydrate, 0.02% (w / vol) zinc chloride, 0.01% (w / vol) iron (III) chloride hexahydrate, 0.004% (w / vol) copper (II) pentahydrate , 0.0008% (w / vol) of manganese chloride tetrahydrate, 0.0006% (w / vol) of boric acid and 0.0026% (w / vol) of hexamonium heptamolybdate tetrahydrate in distilled water, and 100 mL This mixture was placed in an Erlenmeyer flask with 500-mL protrusions, then sterilized in an autoclave at 121 ° C for 15 minutes. After allowing the mixture to cool, PE-M and Tween 80, each having been autoclaved at 121 ° C for 15 minutes separately from the mixture, were individually added to the mixture at 0.01% (w / vol). To this culture medium, Trichoderma reesei PC3-7 was inoculated at 1 * 10 5 cells / mL, and the cells were cultured at 28 ° C for 72 hours with shaking at 180 rpm, to perform the preculture (vibrator) : BIO-VIBRATOR BR-40LF, manufactured by TAITEC CORPORATION).
    Main Culture [0125] A mixture of 5% millhocin (w / vol), 2% glucose (w / vol), 10% (w / vol) cellulose (Avicel), 0.37% tartrate was prepared. ammonium (w / vol),
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    0.14% (w / vol) ammonium sulfate, 0.2% (w / vol) potassium dihydrogen phosphate, 0.03% (w / vol) calcium chloride dihydrate, 0.03% (w / vol) of magnesium sulfate heptahydrate, 0.02% (w / vol) of zinc chloride, 0.01% (w / vol) of iron (III) chloride hexahydrate, 0.004% (w / vol) of pentahydrate sulfate copper (II), 0.0008% (w / vol) manganese chloride tetrahydrate, 0.0006% (w / vol) boric acid and 0.0026% (w / vol) hexammonium heptamolybdate tetrahydrate in water distilled, and 2.5 L of this mixture was placed in a 5-L shaking glass (manufactured by ABLE, DPC-2A), then sterilized in an autoclave at 121 ° C for 15 minutes. After allowing the mixture to cool, PE-M and Tween 80, each having been autoclaved at 121 ° C for 15 minutes separately from the mixture, were added individually to the 0.1% mixture. To the resulting mixture, 250 ml of the pre-culture of Trichoderma reesei PC3-7 preliminarily prepared with a liquid medium through the method described above were inoculated. The cells were cultured at 28 ° C for 87 hours at 300 rpm at an aeration rate of 1 vvm. After centrifugation, the supernatant was subjected to membrane filtration (Stericup-GV, manufactured by Millipore, material: PVDF). To the culture liquid prepared under the conditions already described, β-glucosidase (Novozyme 188) was added in a protein weight ratio of 1/100, and the resulting mixture was used as cellulose based on Trichoderma in the Examples below.
    (Reference Example 4) Cellulase Activity Measurement Method [0126] Cellulase activity was measured and evaluated using the following procedures in terms of four types of degradation activities: 1) Avicel degradation activity; 2) carboxymethyl cellulose (CMC) degradation activity; 3) cellobiose degradation activity; and 4) xylan degradation activity.
    1) Avicel Degradation Activity [0127] To an enzyme liquid (prepared under predetermined conditions), Avicel (Microcrystalline Cellulose, manufactured by Merck) was
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    45/85 added to 1 g / L and the sodium acetate buffer (pH 5.0) was added to 100 mM, then the resulting mixture was allowed to react at 50 ° C for 24 hours. This reaction liquid was prepared in a 1 mL tube, and the reaction was allowed to process by mixing by rotation under the conditions already described. After that, the tube was subjected to centrifugation, and the concentration of glucose in the component supernatant was measured. The measurement of glucose concentration was performed according to the method described in Reference Example 2. The concentration of glucose produced (g / L) was used in the state as the activity value of the Avicel degradation activity.
    2) CMC Degradation Activity [0128] To an enzyme liquid, carboxymethyl cellulose was added at 10 g / L and sodium acetate buffer (pH 5.0) was added at 100 mM, then it was allowed to resulting mixture reacted at 50 ° C for 0.5 hour. This reaction liquid was prepared in a 1 mL tube, and the reaction was allowed to process by mixing by rotation under the conditions already described. After that, the tube was subjected to centrifugation, and the concentration of glucose in the component supernatant was measured. The measurement of glucose concentration was performed according to the method described in Reference Example 2. The concentration of glucose produced (g / L) was used in the state as the activity value of the CMC degradation activity.
    3) Cellobiose Degradation Activity [0129] To an enzyme liquid, cellobiose (Wako Pure Chemical Industries, Ltd.) was added at 500 mg / L and sodium acetate buffer (pH 5.0) was added to 100 mM , then the resulting mixture was allowed to react at 50 ° C for 0.5 hour. This reaction liquid was prepared in a 1 mL tube, and the reaction was allowed to process by mixing by
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    46/85 rotation under the conditions already described. After that, the tube was subjected to centrifugation, and the concentration of glucose in the component supernatant was measured. The measurement of glucose concentration was performed according to the method described in Reference Example 2. The concentration of glucose produced (g / L) was used in the state as the activity value of the cellobiose degradation activity.
    4) Xylan Degradation Activity [0130] To an enzyme liquid, xylan (birch xylan, Wako Pure Chemical Industries, Ltd.) was added at 10 g / L and sodium acetate buffer (pH 5.0) was added to 100 mM, then the resulting mixture was allowed to react at 50 ° C for 4 hours. This reaction liquid was prepared in a 1 mL tube, and the reaction was allowed to process by mixing by rotation under the conditions already described. After that, the tube was subjected to centrifugation, and the xylose concentration in the component supernatant was measured. The measurement of the xylose concentration was performed according to the method described in Reference Example 2. The concentration of xylose produced (g / L) in the state as the activity value of the xylan degradation activity.
    (Example 1) Cellulose Hydrolysis with Trichoderma-Derived Cellulase [0131] The results of primary hydrolysis and secondary hydrolysis in cellulose hydrolysis using Trichoderma-derived cellulase are described in the Examples below. The experiment method was as follows.
    (Step 1: Primary hydrolysis) [0132] To each of the pretreated celluloses 1 to 3 (1 g each), distilled water was added, and 10 mg of cellulase derived from Trichoderma were added, then distilled water was added, so that the total weight was 10 g. In addition, diluted sulfuric acid or dilute caustic soda was added to adjust the pH of the composition to a value in the
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    47/85
    4.5 to 5.3. The composition after pH adjustment was transferred to a side arm test tube (φ30 NS14 / 23, manufactured by Tokyo Rikakikai Co., Ltd.), and the composition was transferred to a side arm reactor (φ30 NS14 / 23 , manufactured by Tokyo Rikakikai Co., Ltd.), then hydrolysis was performed at 50 ° C for 24 hours with incubation and agitation (compact mechanical stirrer CPS-1000, manufactured by Tokyo Rikakikai Co., Ltd., conversion adapter , inlet for feeding with a three-way regulating valve, incubator MG-2200). The hydrolyzate was subjected to solid-liquid separation by centrifugation (3000 G, 10 minutes), and, thus, the hydrolyzate was separated into primary sugar liquid (6 mL) and solids. The concentrations of xylose and glucose in the obtained primary sugar liquid were measured by the methods described in Reference Example 2. The sugar yield (mg) in the primary sugar liquid was calculated according to the equation below. Table 2 shows a summary of the results.
    [0133] Sugar yield (mg) in the primary sugar liquid = {sugar concentration (g / L) after 24 hours of incubation - sugar concentration (g / L) after 0 hour of incubation} χ 6 (mL )
    Table 2
    Sugar concentration (g / L) Sugar yield (mg) (24 h, 5 mL) 0 h 6 h 24 h Pretreated cellulose 1 G1c 0 18 36 216 Xy1 0 6 15 90 Pretreated cellulose 2 G1c 0 27 46 276 Xy1 0 6 8 48 Pretreated cellulose 3 G1c 0 28 35 210 Xy1 0 4 7 42
    (Step 2: Secondary Hydrolysis) [0134] To the solids obtained in Step 1, distilled water was added so that the total weight was 10 g. Additionally, added
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    48/85 if diluted sulfuric acid or diluted caustic soda to adjust the pH of the composition to a value in the range of 4.5 to 5.3. The composition was transferred to a side arm test tube (φ30 NS14 / 23, manufactured by Tokyo Rikakikai Co., Ltd.), and the composition was transferred to a side arm reactor (φ30 NS14 / 23, manufactured by Tokyo Rikakikai Co., Ltd.), then the secondary hydrolysis was performed at 50 ° C for 1 hour with incubation and agitation (compact mechanical stirrer CPS-1000, manufactured by Tokyo Rikakikai Co., Ltd., conversion adapter, feed inlet with a three-way regulating valve, incubator MG-2200). The hydrolyzate was subjected to solid-liquid separation by centrifugation (3000 G, 10 minutes), and, thus, the hydrolyzate was separated into secondary sugar liquid (6 mL) and solids. The concentrations of xylose and glucose in the secondary sugar liquid obtained were measured by the methods described in Reference Example 2. The sugar yield (mg) in the secondary sugar liquid was calculated according to the equation below.
    [0135] Sugar yield (mg) in the secondary sugar liquid = {sugar concentration (g / L) after 1 hour of incubation sugar concentration (g / L) after 0 hour of incubation} χ 6 (mL) [0136 ] Table 3 shows a summary of the results. It was found that, although no additional enzyme was added in the secondary hydrolysis, an hour of incubation caused an increase in the sugar concentration. That is, it was observed that the secondary hydrolysis occurred only with the primary sugar liquid that remained in the solids and / or in the enzyme adsorbed to the solids.
    Table 3
    Sugar concentration (g / L) Sugar yield (mg) (1 h, 6 ml) 0 h 1 h Pretreated cellulose 1 G1c 16 20 24 Xy1 6 8 12
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    Sugar concentration (g / L) Sugar yield (mg) (1 h, Pretreated cellulose 2 G1c 12 16 24 Xy1 1 2 6 Pretreated cellulose 3 G1c 11 16 30 Xy1 1 2 6
    (Step 3: Carbohydrase Recovery) [0137] The primary sugar liquid (6mL) obtained in the primary hydrolysis in Step 1 and the secondary sugar liquid (6mL) obtained in the secondary hydrolysis in Step 2 were mixed together, and the carbohydrase was recovered from the resulting solution.
    [0138] The above solution was filtered through an ultrafiltration membrane that has a cutting molecular weight of 10,000 (VIVASPIN 20, manufactured by Sartorius stedim biotech, material: PES) by centrifugation at 4500 G until the membrane fraction was reduced to 1 ml. To the membrane fraction, 10 ml of distilled water were added, and the resulting mixture was centrifuged again at 4500 G until the membrane fraction was reduced to 1 ml. After that, the enzyme was recovered from the membrane fraction. The protein concentration of the recovered enzyme was assessed with the BCA measurement kit (Protein Assay Reagent BCA kit, manufactured by PIERCE), using bovine albumin (2 mg / mL) as a standard sample, by measuring the absorbance at 562 nm to perform colorimetry. The concentration of the enzyme that could be recovered (mg / mL) was multiplied by 1 mL, which was the amount of the solution in the membrane fraction, to calculate the amount of carbohydrate that could be recovered. As a result, as shown in Table 4, it was found that the enzyme could be recovered in an amount of 1.6 to 2.6 mg.
    Table 4
    Amount of enzyme recovered (mg) 50 ° C Pretreated cellulose 1 1.6
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    Amount of enzyme recovered (mg) 50 ° C Pretreated cellulose 2 2.6 Pretreated cellulose 3 2.0
    (Example 2) Influences of Reaction Temperature on Sugar Yield / Amount of Enzyme Recovered in Secondary Hydrolysis [0139] Secondary hydrolysis was carried out in the same way as Step 2 of Example 1, except for the reaction temperature, which was regulated in range of 25 ° C to 90 ° C. The sugar yield (glucose, g) in the secondary sugar liquid (6 mL) occurred as shown in Table 5. It was found that, in the secondary hydrolysis of the present invention, the sugar yield is the highest, in the range of 40 ° C to 60 ° C, that is, at the optimum reaction temperature of Trichoderma-derived cellulase.
    Table 5
    25 ° C 35 ° C 40 ° C 50 ° C 55 ° C 60 ° C 65 ° C 70 ° C 80 ° C 90 ° C Pretreated cellulose 1 1 3 20 24 22 15 0 0 0 0 Pretreated cellulose 2 2 3 17 24 20 6 0 0 0 0 Pretreated cellulose 3 0 1 20 30 27 10 0 0 0 0
    [0140] Additionally, secondary hydrolysis was performed in the same way as Step 2 of Example 1, except for the reaction temperature, which was regulated in the range of 25 ° C to 90 ° C, and the recovery of carbohydrase in Step 3 was performed , then the amount of carbohydrate that could be recovered was calculated. Consequently, as shown in Table 6, it was found that the
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    51/85 amount of recovered carbohydrase increases in reaction temperatures in the range of 40 to 60 ° C during secondary hydrolysis. It was also possible to confirm that a more preferable temperature during secondary hydrolysis is 50 ° C.
    Table 6
    25 ° C 35 ° C 40 ° C 50 ° C 55 ° C 60 ° C 65 ° C 70 ° C 80 ° C 90 ° C Pretreated cellulose 1 0 0 1.0 1.6 1.4 1.4 0 0 0 0 Pretreated cellulose 2 0 0 1.6 2.6 2.1 1.3 0 0 0 0 Pretreated cellulose 3 0 0 1.4 2.0 1.5 1.3 0 0 0 0
    (Example 3) Analysis of Recovered Enzyme [0141] The recovered enzyme obtained in Step 3 of Example 1 (pretreated cellulose 2) was electrophoresed by SDS-PAGE to analyze the components of the recovered enzyme. First, an equal amount of a sample treatment buffer (Ez Apply, manufactured by ATTO Corporation) was mixed with the recovered enzyme, and the resulting mixture was treated at 100 ° C for 10 minutes. At 15% gel for electrophoresis (e-PAGEL, manufactured by ATTO Corporation), 5 pL of the treated sample was applied, and electrophoresis was performed (40 mA, 30 minutes). The gel was removed and marked with bright Coomassie blue (Bio-safe CBB, manufactured by Bio-Rad Laboratories), then discolored with distilled water. The result obtained by gel staining after electrophoresis is shown in Figure 9. It is possible to confirm that, in the range of 40 ° C to 60 ° C, especially cellobiohydrolase among the components of cellulase derived from Trichoderma is contained as a recovered enzyme component .
    (Example 4) Relationship between Reaction Time and Sugar Yield / Amount of Enzyme Recovered in Secondary Hydrolysis [0142] Secondary hydrolysis was performed in the same way as Step 2 of Example 1, except for the reaction time, which was regulated in the range
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    52/85 from 0 to 720 minutes. The yield of sugar (glucose, g) in the secondary sugar liquid (6 ml) occurred as shown in Table 7. It was found that a sufficient amount of sugar can be produced by performing the secondary hydrolysis of the present invention for not less than 5 minutes. On the other hand, the amount of the recovered enzyme was not changed in the range greater than 180 minutes.
    Table 7
    0 1 5 10 30 60 120 180 360 720 Pretreated cellulose 1 0 1 2 6 18 24 30 35 36 36 Pretreated cellulose 2 0 1 4 6 14 24 29 34 34 35 Pretreated cellulose 3 0 1 3 8 20 30 38 42 42 42
    [0143] Subsequently, secondary hydrolysis was carried out in the same way as Step 2 of Example 1, except for the reaction time, which was regulated in the range of 0 to 720 minutes, and the recovery of carbohydrase in Step 3 was performed, then the amount of carbohydrate that could be recovered was calculated. Consequently, as shown in Table 8, it was found that a sufficient amount of the enzyme can be recovered by performing the secondary hydrolysis of the present invention for at least 5 minutes. On the other hand, the amount of the recovered enzyme was not changed in the range greater than 180 minutes.
    Table 8
    0 1 5 10 30 60 120 180 360 720 Pretreated cellulose 1 0 0.1 0.3 0.5 1.2 1.6 1.9 2.4 2.4 2.6 Pretreated cellulose 2 0 0.1 0.5 1.1 1.8 2.6 3.0 3.5 3.6 3.7
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    0 1 5 10 30 60 120 180 360 720 Pretreated cellulose 3 0 0 0.5 0.8 1.5 2.0 2.4 2.8 3.0 3.1
    (Example 5) Relationship between pH and Amount of Enzyme Recovered in Hydrolysis
    Secondary [0144] Step 2 of Example 1 was performed at 50 ° C after adjusting the pH to a value in the range of 4.5 to 5.3 with diluted sulfuric acid or diluted caustic soda. However, in the present Example, the pH was adjusted with dilution buffers to various values in the range of 3 to 10, and secondary hydrolysis was carried out at 50 ° C. As a buffer, 2 mM sodium acetate buffer was used for pHs in the range 3 to 8, while the 2 mM glycine-hydroxide buffer was used for pHs in the range 9 to 10. The experiment was carried out in the same way than in Example 1, except for the pH adjustment. As a result, as shown in Table 9, it was found that the amount of the recovered enzyme can be increased by performing secondary hydrolysis at a pH in the range of 6.0 to 8.0.
    Table 9
    3 4 5 6 7 8 9 10 Pretreated pulp 1 0 1.2 1.6 3.0 3.4 3.4 2.0 2.0 Pretreated pulp 2 0 1.8 2.6 3.5 3.8 4.0 2.1 2.1 Pretreated pulp 3 0 1.5 2.0 3.2 3.5 3.2 1.0 1.0
    (Example 6) Relationship between Quantity of Added Nonionic Surfactant and
    Amount of Enzyme Recovered in Secondary Hydrolysis [0145] Pluronic F-68 (manufactured by BASF) was used as a nonionic surfactant. The secondary hydrolysis was carried out in the same way as the Step of Example 1, except for Pluronic F-68, which was added so that its
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    54/85 final concentration was in the range of 0.01 to 5%. The same method as in Example 1, except for the addition of a nonionic surfactant. As a result, as shown in Table 10, the amount of the recovered enzyme could be increased by adding the nonionic surfactant in concentrations ranging from 0.05% to 2%.
    Table 10
    0.01% 0.05% 0.1% 0.25% 0.5% 1% 2% 5% Pretreated cellulose 1 1.6 1.8 2.1 2.3 2.5 2.9 3.3 3.3 Pretreated cellulose 2 2.7 3.0 3.2 3.6 3.8 4.1 4.2 4.2 Pretreated cellulose 3 2.0 2.1 2.4 2.6 2.9 3.4 3.7 3.8
    (Reference Example 4) Mass Production of Primary Hydrolyzate [0146] For mass production of the primary hydrolyzate, 20 g of cellulase derived from Trichoderma were added to pretreated cellulose 3 (2 kg), and distilled water was additionally added so that the total weight was 20 kg. Additionally, the pH of the composition was adjusted to a value in the range of
    4.5 to 5.3 with diluted sulfuric acid or diluted caustic soda. While the liquid was incubated so that a liquid temperature of 45 to 50 ° C was maintained, and the diluted sulfuric acid and / or the diluted caustic soda was / were added to the liquid so that the pH was maintained in the range of 4.5 at 5.3, the enzyme was allowed to react with pretreated cellulose 3 for 24 hours (the liquid obtained by the reaction is hereinafter called the pasty liquid of enzymatic saccharification).
    (Comparative Example 1) Solid-Liquid Separation Through Filtration Using Filter Press (Solid-Liquid Separation After Primary Hydrolysis) [0147] Using 10 L of the pasty liquid from enzymatic saccharification obtained in Reference Example 4, filtration using a filter press was performed using the following procedure. For filtration using filter
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    55/85 press, a compact filter press apparatus (filter press MO-4, manufactured by Yabuta Industries Co., Ltd.) was used. As a filter cloth, a woven polyester cloth (T2731C, manufactured by Yabuta Industries Co., Ltd.) was used. After feeding 10 L of the pasty liquid of enzymatic saccharification into the compact tank, the liquid inlet was opened to slowly feed the pasty liquid of enzymatic saccharification into the filtration chamber using an air pump (66053-3EB, manufactured by Taiyo International Corporation) by aeration with compressed air through the bottom. As the filtrate obtained after 1 minute of the operation remained turbid, the filtrate was returned to the compact tank. The actuation pressure of the air pump was gradually increased to increase the pressure in the filtration chamber, and the injection step was continued until the filtrate was obtained. The maximum injection pressure this time was 0.15 MPa. The time required for the injection step above was 30 minutes.
    [0148] Subsequently, a compression step was performed by expanding the diaphragm attached to the filtration chamber. The compression pressure was gradually raised to 0.5 MPa and the device rested for about 30 minutes to recover the filtrate. The time required for the compression step above was 40 minutes.
    [0149] After the compression step was completed, the pressure in the diaphragm and the tank was released, and the obtained solids were collected. The total amount of filtrate obtained was 9.0 L. The remaining liquid component was lost due to the dead volume of the device. Based on the results of the sugar concentrations in the filtrate obtained, that is, the primary sugar liquid of Comparative Example 1, the glucose concentration was 35 g / L and the xylose concentration was 7 g / L. That is, in Comparative Example 1, the glucose yield was 315 g and the xylose yield was 63 g (Table 11).
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    56/85 (Comparative Example 2) Solid-Liquid Separation Through Filtration Using Filter Presses (Solid-Liquid Separation and Solid Wash After Primary Hydrolysis) [0150] Using 10 L of the pasty liquid from enzymatic saccharification obtained in Reference Example 4 , filtration using a filter press was performed using the following procedure. Filtration using a filter press was performed using the same apparatus as in Comparative Example 1, and the operating conditions were the same as the conditions in Reference Example 1 until the injection stage. In the compression stage, the compression pressure was increased to 0.2 MPa, and the filtrate was collected for about 5 minutes. The amount of filtrate collected this time was 8 L. Based on the results of the sugar concentrations in the filtrate obtained, that is, the primary sugar liquid in Comparative Example 2, the glucose concentration was 35 g / L and the concentration xylose was 7 g / L. Therefore, in primary hydrolysis, the glucose yield (sugar yield in the primary sugar liquid) was 280 g and the xylose yield was 56 g (Table 11).
    [0151] Subsequently, the remaining solids in the filtration chamber using a filter press were washed with water using the following procedure. We must realize that this operation is not secondary hydrolysis.
    [0152] To the tank, 2.5 L of distilled water at 18 ° C were added, and the injection step was carried out at an injection pressure of 0.2 MPa until no more filtrate was produced. After that, a compression step was performed by expanding the diaphragm attached to the filtration chamber. The compression pressure was gradually raised to 0.5 MPa and the device rested for about 30 minutes to recover the filtrate. The time required for the above compression step was 35 minutes. After the compression step was completed, the pressure in the diaphragm and the tank was released, and the solids obtained were collected. The total amount of filtrate
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    57/85 obtained was 3.5 L. The remaining liquid component was lost due to the dead volume of the device. Based on the results of the sugar concentrations in the filtrate obtained, that is, the secondary sugar liquid of Comparative Example 2, the glucose concentration was 14 g / L and the xylose concentration was 3.1 g / L. Therefore, this time the glucose yield was 49 g and the xylose yield was 11 g (Table 11).
    Table 11
    Primary hydrolysis (g) Solid washing liquid (g) Total sugar yield (g) Comparative Example 1 G1c 315 0 315 Xy1 63 0 63 Comparative Example 2 G1c 280 49 329 Xy1 56 11 67
    (Example 6) Secondary Hydrolysis in the Filtration Chamber using Filter Press [0153] Using 10 L of the pasty liquid of the enzymatic saccharification obtained in Reference Example 4, the solid-liquid separation and the secondary hydrolysis were carried out using the following procedure.
    [0154] Filtration using a filter press was performed using the same apparatus as in Comparative Example 2 under the same operating conditions. That is, the amount of the filtrate that could be collected was 8 L. Based on the results of the sugar concentrations in the filtrate obtained, that is, in the primary sugar liquid of Example 2, the glucose concentration was 35 g / L and the xylose concentration was 7 g / L. Therefore, the glucose yield was 280 g and the xylose yield was 56 g this time. Table 12 shows a summary of the results as the sugar yield in the primary sugar liquid of Example 6.
    [0155] Subsequently, secondary hydrolysis in the filtration chamber using a filter press was performed using the following procedure.
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    58/85 [0156] During the compression stage, the
    2.5 L of distilled water in the tank. After that, the tank was heated using a rubber heater until the temperature of the distilled water reached 50 ° C. After heating, warm water at 50 ° C was fed to the filtration chamber in the same way as the pasty liquid. All filtrate obtained was returned to the tank to allow circulation. This circulation was continued until 1 hour after the start of the filtrate production. Based on the result of the filtrate temperature measurement, the filtrate was at 40 ° C after 10 minutes, and remained constant at 45 ° C after 15 minutes and thereafter. The injection pressure was kept constant at 0.15 MPa. After 120 minutes, the filtrate side operation was diverted for removal, instead of circulating to the tank. The injection step was continued at an injection pressure of 0.2 MPa until no more filtrate was produced. Subsequently, a compression step was performed by expanding the diaphragm attached to the filtration chamber. The compression pressure was gradually raised to 0.5 MPa and the device rested for about 30 minutes to recover the filtrate. The time required for the above compression step was 35 minutes. After the compression step was completed, the pressure in the diaphragm and the tank was released, and the solids obtained were collected. The total amount of filtrate obtained was 3.5 L. The remaining liquid component was lost due to the dead volume of the device. Based on the results of the sugar concentrations in the filtrate obtained, the glucose concentration was 28 g / L and the xylose concentration was 5 g / L. Therefore, the glucose yield was 98 g and the xylose yield was 17.5 g this time. Table 12 shows a summary of the results as the sugar yield in the secondary sugar liquid obtained by the secondary hydrolysis and the solid-liquid separation in Example 6.
    Table 12
    Primary hydrolysis (g) Secondary hydrolysis (g Total sugar yield (g)
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    Primary hydrolysis (g) Secondary hydrolysis(g) Total sugar yield (g) Example6 G1c 280 98 378 Xy1 56 17.5 73.5
    [0157] Comparison of the results of Comparative Example 1 and Comparative Example 2, in which secondary hydrolysis was not performed, with the results of Example 6 shown in Table 12 revealed that the sugar yield can be greatly increased by performing the secondary hydrolysis of the present invention.
    (Example 7) Amount of Carbohydrase Recovery Obtained by Secondary Hydrolysis in the Filtration Chamber using Filter Presses [0158] The primary sugar liquid (6 mL) and the secondary sugar liquid (6 mL) obtained in Example 6 were mixed together, and carbohydrase was recovered from the resulting sugar liquid. For comparison, the primary sugar liquid (6mL) and the solid wash liquid (6 mL) obtained in Comparative Example 2 were mixed together, and the carbohydrase was also recovered from the resulting sugar liquid. The sugar liquid was filtered through an ultrafiltration membrane that has a cutting molecular weight of 10,000 (VIVASPIN 20, manufactured by Sartorius stedim biotech, material: PES) by centrifugation at 4500 G until the membrane fraction was reduced to 1 ml. To the membrane fraction, 10 ml of distilled water were added, and the resulting mixture was centrifuged again at 4500 G until the membrane fraction was reduced to 1 ml. After that, the enzyme was recovered from the membrane fraction. The protein concentration of the recovered enzyme was measured with the BCA measurement kit (Protein Assay Reagent BCA kit, manufactured by PIERCE), using bovine albumin (2 mg / mL) as a standard sample, by measuring the absorbance at 562 nm to perform colorimetry. The concentration of the enzyme that could be recovered (mg / mL) was multiplied by 1 mL, which was the amount of the solution in the fraction of the
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    60/85 membrane, to calculate the amount of carbohydrate that could be recovered. As a result, as shown in Table 13, it was possible to confirm that the amount of carbohydrate recovered is increased with the effect produced by the passage of warm water in Example 6 in relation to the amount of enzyme recovered from the sugar liquid of Comparative Example 2.
    Table 13
    Amount of enzyme recovered (mg) 50 ° C Comparative Example 2 1.6 Example 6 3.9
    (Example 8) Carbohydrase Recovery and Reuse [0159] The primary sugar liquid (8 L) and the secondary sugar liquid (3.5 L) obtained in Comparative Example 2 and Example 6 were mixed together, and the carbohydrase was recovered from 11.5 L of each of these resulting mixtures. The recovery of carbohydrase was carried out using a smooth and compact membrane filtration device (Sepa (registered trademark) CF II Med / High Foulant System, manufactured by GE) equipped with a smooth ultrafiltration membrane that has a cutting molecular weight of 10000 (SEPA series PW, manufactured by GE, functional surface material: polyether sulfone). While the operating pressure was controlled so that the flow rate on the feed side was constantly 2.5 L / minute and the membrane flow was constantly 0.1 m / D. 11 L of 11.5 L were filtered. Therefore, 0.5 L of carbohydrase was recovered from the feeding side. Subsequently, the recovered enzyme obtained in an amount of 0.5 L was added to the pre-treated cellulose 3 (1 kg), and distilled water was still added so that the total weight was 10 kg. In addition, diluted sulfuric acid or diluted caustic soda was added to adjust the pH of the
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    61/85 composition at a value in the range of 4.5 to 5.3. While the liquid was incubated so that a liquid temperature of 45 to 50 ° C was maintained, and diluted sulfuric acid and / or diluted caustic soda was / were added to the liquid so that the pH was maintained in the range of 4.5 to 5 , 3, the enzyme was allowed to react with pretreated cellulose 3 for 24 hours. As a result, as shown in Table 14, it was possible to confirm that the sugar yield in the case where the enzyme recovered from the sugar liquids (primary and secondary) in Example 6 was higher than the sugar yield in the case where it was the enzyme recovered from the sugar liquids (primary and secondary) of Comparative Example 2 was used.
    Table 14
    Sugar concentration (g / L)24 h Sugar yield (mg) (24 h, 6 mL) Comparative Example 2 G1c 10 60 Xy1 2 12 Example 6 G1c 27 162 Xy1 4 24
    (Example 9) Concentration of the Primary Sugar liquid and Secondary Sugar liquid with Reverse Osmosis Membrane (RO Membrane) [0160] Individually, the primary sugar liquid and the secondary sugar liquid in Example 6 in an amount of 1 L were concentrated with a reverse osmosis membrane (RO membrane). First, each of them, the primary sugar liquid and the secondary sugar liquid, was pre-filtered through a microfiltration membrane that has a pore size of 0.45 pm. Each of the liquids obtained by processing the membrane in an amount of 1 L was used to perform the concentration with an RO membrane. As an RO membrane, a fully crosslinked aromatic reverse osmosis membrane “UTC80” (manufactured by Toray Industries, Inc.) was used. THE
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    62/85 RO membrane was mounted on a smooth and compact membrane filtration device (Sepa (registered trademark) CF II Med / High Foulant System, manufactured by GE), and the filtration treatment was carried out at a gross temperature of the liquid 25 ° C and a pressure of 3 MPa using a high pressure pump. With this treatment, 0.7 L of the permeate was obtained. The concentrations of xylose and glucose this time were the concentrations shown in Table 15, and it was possible to confirm that the sugar concentrations in the primary sugar liquid and in the secondary sugar liquid may have their concentration increased with an RO membrane.
    Table 15
    Untreated (g / L) After concentration with an RO membrane (g / L) Concentration rate (factor) Primary sugar liquid G1c 35 115 3.3 Xy1 7 23 3.3 Secondary sugar liquid G1c 28 92 3.3 Xy1 5 16 3.2
    (Example 10) Concentration of Primary Sugar liquid and Secondary Sugar liquid with Nanofiltration Membrane (NF Membrane) [0161] The primary sugar liquid and the secondary sugar liquid individually in Example 8 in an amount of 1 L were concentrated with a nanofiltration membrane (NF membrane).
    [0162] First, each of them, the primary sugar liquid and the secondary sugar liquid, were pre-filtered through a microfiltration membrane that has a pore size of 0.45 pm. Each of the liquids obtained by processing on the membrane in an amount of 1 L was used to perform the concentration with an NF membrane. Like the NF membrane, a “UTC60” crosslinked piperazine and polyamide nanofiltration membrane (manufactured by Toray Industries, Inc.) was used. The NC membrane was mounted on a filtration device by
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    63/85 smooth and compact membrane (Sepa (registered trademark) CF II Med / High Foulant System, manufactured by GE), and filtration treatment was carried out at a gross liquid temperature of 25 ° C and a pressure of 3 MPa using a high pressure pump. With this treatment, 0.7 L of the permeate was obtained. The concentrations of xylose and glucose this time were the concentrations shown in Table 16, and it was possible to confirm that the sugar concentrations in the primary sugar liquid and in the secondary sugar liquid may have their concentration increased with an NF membrane.
    Table 16
    Untreated (g / L) After concentration with an NF membrane (g / L) Concentration rate (factor) Primary sugar liquid G1c 35 112 3.2 Xy1 7 20 2.8 Secondary sugar liquid G1c 28 90 3.2 Xy1 5 14 2.8
    (Comparative Example 3) Solid-Liquid Separation by Centrifugation (Solid-Liquid Separation After Primary Hydrolysis) [0163] Using 10 L of the pasty liquid from the enzymatic saccharification obtained in Example 7, centrifugation was performed using the following procedure.
    [0164] Each of the 25 500 mL centrifuge tubes was fed with 400 mL of the enzyme saccharification slurry, and the slurry was subjected to centrifugation at 3000 G for 10 minutes. From each centrifuge tube, 240 mL of the supernatant could be collected, and a total of 6 L of the supernatant, therefore, could be collected from the 25 centrifuge tubes. The remaining 160 mL (4 L in total) of the content in each centrifuge tube was considered to be the solid in the centrifuge tube and collected. Based on the results of the sugar concentrations in the filtrate obtained, that is, the primary sugar liquid in Example 8, the glucose concentration was 35
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    64/85 g / L and the xylose concentration was 7 g / L. Therefore, in the case where the sugar was obtained by centrifugation for solid-liquid separation, the glucose yield (sugar yield in the primary sugar liquid) was 210 g and the xylose yield was 42 g (Table 17).
    (Comparative Example 4) Solid-Liquid Separation by Centrifugation (Solid-Liquid Separation and Washing of Solids After Primary Hydrolysis) [0165] Using 10 L of the pasty liquid of the enzymatic saccharification obtained in Example 7, centrifugation was performed through the procedure a follow.
    [0166] Each of the 25 500 mL centrifuge tubes was fed with 400 mL of the enzyme saccharification slurry, and the slurry was subjected to centrifugation as in Comparative Example 3. A total of 6 L of the supernatant could be collected .
    [0167] The remaining 160 mL (4 L in total) of the content in each centrifuge tube was considered as the solid in the centrifuge tube and collected. Based on the results of the sugar concentrations in the filtrate obtained, that is, the primary sugar liquid of Example 8, the glucose concentration was 35 g / L and the xylose concentration was 7 g / L. Therefore, in the case where the sugar was obtained by centrifugation for solid-liquid separation, the glucose yield (sugar yield in the primary sugar liquid) was 210 g and the xylose yield was 42 g (Table 17).
    [0168] Subsequently, the remaining solids were washed with water using the following procedure. We must realize that this operation is not secondary hydrolysis.
    [0169] To the solids collected by centrifugation, water was added, and the centrifugation was performed again to perform the solid-liquid separation, thus collecting the sugar liquid. To the precipitated component in
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    65/85 a quantity of 160 mL remaining in each of the 25 centrifuge tubes, 100 mL of distilled water were added. The temperature of the distilled water this time was 18 ° C. After adding water, the centrifuge tubes were slightly rotated to mix their contents, and centrifuged again at 3000 G for 10 minutes. After that, 150 mL of the supernatant could be collected from each centrifuge tube. That is, a total of 3.75 L of the supernatant could be collected from the 25 centrifuge tubes. Based on the results of the sugar concentrations in the obtained supernatant, that is, the secondary sugar liquid of Comparative Example 4, the glucose concentration was 21 g / L and the xylose concentration was 4 g / L. Therefore, this time, the glucose yield was 79 g and the xylose yield was 15 g (Table 17).
    Table 17
    Primary hydrolysis (g) Solid washing liquid (g) Total sugar yield (g) Comparative Example 3 G1c 210 0 210 Xy1 42 0 42 Comparative Example 4 G1c 210 79 289 Xy1 42 15 57
    (Example 11) Solid-Liquid Separation by Centrifugation (Hydrolysis
    Primary and Secondary Hydrolysis) [0170] Using 10 L of the pasty liquid of the enzymatic saccharification obtained in Reference Example 4, centrifugation was performed using the following procedure.
    [0171] Each of the 25 500 mL centrifuge tubes was fed with 400 mL of the enzyme saccharification slurry, and the slurry was subjected to centrifugation as in Comparative Example 3. A total of 6 L of the supernatant could be collected .
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    66/85 [0172] Subsequently, the secondary hydrolysis of the remaining solids was carried out using the following procedure. Each of the 25 500 ml centrifuge tubes was fed with 400 ml of the enzyme saccharification slurry, and the slurry was subjected to centrifugation at 3000 G for 10 minutes. After centrifugation, 240 mL of the supernatant could be collected from each centrifuge tube. That is, a total of 6 L of the supernatant could be collected from the 25 centrifuge tubes. The remaining 160 mL (4 L in total) of the content in each centrifuge tube was considered as the precipitated component in the centrifuge tube and collected. Based on the results of the sugar concentrations in the filtrate obtained, that is, the primary sugar liquid of Example 11, the glucose concentration was 35 g / L and the xylose concentration was 7 g / L. Therefore, in the case where the sugar was obtained by centrifugation for solid-liquid separation, the glucose yield (sugar yield in the primary sugar liquid) was 210 g and the xylose yield was 42 g. These results are summarized in Table 17 as the sugar yield in the case of primary hydrolysis (yield of sugar in the primary sugar liquid).
    [0173] To the precipitated component collected by centrifugation, water was added, and the centrifugation was performed again to perform the solid-liquid separation, thus collecting the sugar liquid. To the precipitated component in an amount of 160 ml remaining in each of the 25 centrifuge tubes, 100 ml of distilled water preheated to 50 ° C were added. After that, the centrifuge tubes were slightly rotated to mix their contents, and left in an incubator maintained at 50 ° C for 1 hour. Subsequently, the centrifuge tubes were slightly rotated to mix their contents, and centrifuged again at 3000 G for 10 minutes. In this way, 150 mL of the supernatant could be collected from each centrifuge tube. That is, a total of 3.75 L of the supernatant could be
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    67/85 collected from 25 centrifuge tubes. Based on the results of the sugar concentrations in the obtained supernatant, i.e., the secondary sugar liquid of Example 11, the glucose concentration was 29 g / L and the xylose concentration was 5 g / L. Therefore, this time the glucose yield was 109 g and the xylose yield was 19 g (Table 18).
    Table 18
    Primary hydrolysis (g) Secondary hydrolysis(g) Total sugar yield (g) Example11 G1c 210 109 319 Xy1 42 19 61
    [0174] Based on the comparison of the results of Comparative Example 3 and Comparative Example 4 shown in Table 17, in which secondary hydrolysis was not performed, with the results of Example 11 shown in Table 18, it was found that the sugar yield can be greatly increased by carrying out the secondary hydrolysis of the present invention. However, based on the comparison of the results of Example 11 with the results of Example 6, in which the solid-liquid separation was carried out through filtration using a filter press, it was found that the sugar yield is lower in cases where the Separation of the liquid is carried out by centrifugation, as observed by the comparison between Table 12 and Table 18. The reason for this is that a higher efficiency of sugar recovery through solid-liquid separation is obtained by filtration using a filter press of the than by centrifugation.
    (Example 12) Enzyme Cellulose Degradation Activity Recovered in Secondary Hydrolysis [0175] Secondary hydrolysis was carried out under the conditions of Example 1 (pretreated cellulose 3, 50 ° C), Example 2 (pretreated cellulose 3, 25 ° C) and Example 5 (pretreated cellulose 3, Pluronic F68: 0.1%, 0.25%, 0.5%, 1%, 2%), and the cellulase activity of each recovered enzyme was measured by method in
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    Reference Example 4. Table 19 shows a summary of the activity values represented as the relative values calculated using as standard the activity of the enzyme recovered after secondary hydrolysis under the conditions of Example 1 (50 ° C) (activity = 1). Xylan degradation activity could not be detected for some conditions, and in such cases it was represented as ND (not detected). Since the xylan degradation activity was also ND for Example 1, which was used as the standard, the xylan degradation activity observed with Pluronic F68: 0.5% for Example 5 was set to 1 to describe the relative activities .
    Table 19
    ^^^ Activity Conditions' ^ - ^^ pH Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan degradation activity Example 1 (50 ° C) 5.0 1 1 1 ND Example 2 (25 ° C) 5.0 ND ND ND ND Example 5 (50 ° C, Pluronic F-68) 0.1% 5.0 1.8 1.2 1.2 ND 0.25% 5.0 1.9 1.4 1.5 ND 0.5% 5.0 2.3 1.4 1.5 1 1% 5.0 3.0 1.4 1.7 1.3 2% 5.0 3.2 1.5 1.9 1.5
    [0176] No cellulase activity was observed for the recovered enzyme for conditions at 25 ° C (Example 2). On the other hand, all cellulase activities were higher in the enzymes recovered for the conditions at 50 ° C, especially at 50 ° C in the presence of the nonionic surfactant (Pluronic F-68) (Example 5).
    (Comparative Example 5) Additive Effects on Enzyme Recovery using Cationic and Anionic Surfactants [0177] As a compound to be added for secondary hydrolysis, sodium lauryl sulfate (SDS), which is an anionic surfactant, or 1% chloride, was used benzalkonium, which is a cationic surfactant. THE
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    69/85 secondary hydrolysis was carried out as in Example 1 (pretreated cellulose 3), except for the surfactant, which was added so that its final concentration was 1%. The recovered enzyme was obtained using the same procedure as in Example 1 and the cellulase activity of the recovered enzyme was measured, but the activity could not be detected for any of the conditions. Therefore, it was possible to confirm that, to recover the enzyme and at the same time maintain its activity, neither a cationic surfactant nor an anionic surfactant can be used.
    (Example 13) Relationship between the Type of Inorganic Salt in Secondary Hydrolysis and Recovered Enzyme Activity [0178] As compounds to be added for secondary hydrolysis, various inorganic salts (sodium chloride, sodium acetate, sodium sulfate, sodium, sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium chloride, dipotassium hydrogen phosphate, ammonium chloride, magnesium chloride, magnesium sulfate and calcium chloride) were used. Secondary hydrolysis was carried out as in Example 1 (pretreated cellulose 3), except that the inorganic salts were added so that their final concentration was 1%. The pH after the addition was adjusted with sodium hydroxide or hydrochloric acid to a value in the range of 5.5 to 6.0. The experiment was carried out under the same conditions as in Example 1 (at 50 ° C, for 1 hour) except for the addition of an inorganic salt.
    [0179] Table 20 shows a summary of the activity values represented as relative values calculated using as standard the activity of the enzyme recovered after secondary hydrolysis under the conditions of Example 1 (pretreated cellulose 3, 50 ° C) (activity = 1). Xylan degradation activity could not be detected for some conditions, and was represented as ND (not detected) in such cases. Since the xylan degradation activity was ND also for Example 1, which was
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    70/85 used as the standard, the xylan degradation activity observed in the case of dipotassium hydrogen phosphate was set to 1 to describe the relative activities.
    Table 20
    ^ '^^ ActivityConditions ^^^^ PH Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan degradation activity Example 1 5.0 1 (Default) 1 (Default) 1 (Default) ND Sodium chloride 5.8 15 3.3 2.5 3 Sodium acetate 5.9 3.7 2.8 1.5 1.5 Sodium sulphate 6.0 12 3.3 1.8 1.5 Sodium hydrogen sulfate 6.0 11 3.0 1.4 1.1 Sodium dihydrogen phosphate 5.9 4.5 3.0 1.4 1.7 Sodium hydrogen phosphate 6.0 5 3.3 1.9 2.5 Potassium chloride 6.1 5 3.3 1.9 2.5 Dipotassium hydrogen phosphate 6.0 3 2 1.4 1 (Default) Ammonium chloride 6.0 7 3.3 2.2 3.3 Magnesium chloride 5.8 30 3.3 4.3 5.5 Magnesium sulfate 5.7 15 3.3 2.4 5.5 Calcium chloride 5.6 ND ND ND ND Calcium sulfate - Not feasible Not feasible Not feasible Not feasible Calcium hydrogen carbonate - Not feasible Not feasible Not feasible Not feasible
    [0180] Consequently, as shown in Table 20, it was found that the amount of enzyme recovered tends to be greater in cases where an inorganic salt has been added than in cases where no inorganic salt has been added. However, it was found that the recovered enzyme shows weak activity in the case of calcium chloride, which is an inorganic calcium salt. Calcium sulfate and calcium hydrogen carbonate, which are also calcium salts, were studied in the same way, but the experiment could not be carried out, because these salts could not be dissolved at the predetermined concentration (1%, final concentration) due to
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    71/85 its low solubility in water. Therefore, it has been found that inorganic calcium salts, among inorganic salts, are not suitable as a compound that will be added for the secondary hydrolysis of the present invention.
    (Example 14) Influence of Concentration of Added Inorganic Salt on Secondary Hydrolysis [0181] The influence of the concentration of added inorganic salt was confirmed using sodium chloride, which is a sodium salt. Using the same procedure as in Example 13, secondary hydrolysis was carried out for sodium chloride concentrations of 0%, 0.1%, 0.5%, 1% and 5%, and the cellulose degradation activity of the recovered enzyme was measured in the same way as in Reference Example 4. Table 21 shows a summary of the activity values represented as relative values calculated using as standard the activity of the enzyme recovered after secondary hydrolysis under the conditions of Example 1 (50 ° C ) (activity = 1). Xylan degradation activity could not be detected for some conditions, and was represented as ND (not detected) in such cases. As the xylan degradation activity was also ND for Example 1, which was used as the standard, the xylan degradation activity observed with 0.5% sodium chloride was set to 1 to describe the relative activities.
    Table 21
    ^ '' '^ ActivityConditions Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan activity and degradation Example 1 1 (Default) 1 (Default) 1 (Default) ND 0.1% 2.8 1.3 1.5 ND 0.5% 7.8 2.4 1.8 1 (Default) 1% (Example 13) 15 3.3 2.5 3 5% 20 4.2 2.5 8
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    72/85 [0182] As shown in Table 21, it was found that the addition of sodium chloride as an inorganic salt even at a minimum concentration of 0.1% increases the Avicel degradation activity of the recovered enzyme, and the addition sodium chloride in a concentration of not less than 0.5% greatly increases all degradation activities compared to the case where sodium chloride was not added.
    (Example 15) Relationship between the addition of inorganic salt and the temperature of the secondary hydrolysis [0183] The secondary hydrolysis was carried out in the presence of 1% sodium chloride at temperatures of 30 ° C, 40 ° C, 50 ° C and 60 ° C, and the activity of the recovered enzyme was measured. Table 22 shows a summary of the activity values represented as relative values calculated using as a standard the activity of the enzyme recovered after secondary hydrolysis under the conditions of Example 1 (pretreated cellulose 3, 50 ° C) (activity = 1) . Xylan degradation activity could not be detected for some conditions, and was represented as ND (not detected) in such cases. Since the xylan degradation activity was also ND for Example 1, which was used as the standard, the xylan degradation activity observed with 1% sodium chloride at 60 ° C was set to 1 to describe the relative activities.
    Table 22
    ^ '' '' '' -.- Activity Conditions' '··' ^^ Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan degradation activity Example 1 1 (Default) 1 (Default) 1 (Default) ND 30 ° C 4.4 3.0 2.0 3 40 ° C 7.4 3.3 2.1 3.2 50 ° C (Example 13) 15 3.3 2.5 3 60x: 2.8 3.0 2.1 1 (Default)
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    73/85 [0184] As shown in Table 22, it was found that, in the presence of sodium chloride, especially the degradation activity of Avicel tends to increase as the temperature approaches 50 ° C, but the activity decreases at 60 ° C. In terms of CMC degradation activity and cellobiose degradation activity, no temperature dependent increase in the amount of recovery could be observed, and the degradation activities did not decrease even at a temperature above 60 ° C. In terms of the xylan degradation activity, it was found that the degradation activity of the recovered enzyme is higher at temperatures ranging from 30 ° C to 50 ° C, but the activity is drastically reduced to 60 ° C.
    (Example 16) Use of Sea Water as Inorganic Salt in Secondary Hydrolysis [0185] In Examples 13 to 15, it was possible to confirm that adding an inorganic salt can increase the amount and activity of the recovered enzyme. In view of this, a study was carried out to verify whether “sea water”, which is an aqueous solution containing inorganic salts, can be used as an alternative. As seawater, seawater collected near the fishing port of Misaki in Kanagawa (pH 8.3; amount of dissolved solid, 3.2%) was used. The pH of sea water was adjusted with sulfuric acid to pH 6.5 (addition of 24 mg of sulfuric acid per 1 L of sea water), pH 5.0 (addition of 50 mg of sulfuric acid per 1 L of seawater) or pH 3.8 (addition of 100 mg of sulfuric acid per 1 L of seawater). In terms of the amount of sea water for secondary hydrolysis, sea water was added to the solids so that its final concentration was 50% (dilution rate, 1/2). Secondary hydrolysis was carried out at 50 ° C for 1 hour. Table 23 shows the activities of the enzyme that could be recovered. The table summarizes the activity values represented as relative values calculated using as standard the activity of the enzyme recovered afterwards
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    74/85 to secondary hydrolysis under the conditions of Example 1 (cellulose, pretreated product 3, 50 ° C) (activity = 1). Xylan degradation activity could not be detected for some conditions, and was represented as ND (not detected) in such cases. As the xylan degradation activity was also ND for Example 1 (cellulose, pretreated product 3, 50 ° C), which was used as the standard, the xylan degradation activity observed in the case of sea water at pH 3.8 was set to 1 to describe the relative activities.
    Table 23
    ^^ - .. ^ ActivityConditions ^ · '.. ^^ Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan degradation activity Example 1 1 (Standard) · 1 (Default) 1 (Default) ND Sea water pH 8.3 12 3.0 2.3 3 pH 6.5 15 3.3 2.3 3.2 pH 5.0 18 3.3 2.5 3 pH 3.8 2.2 2.0 2.2 1 (Default)
    [0186] As shown in Table 23, it was possible to confirm that, even in cases where sea water was used as the inorganic salt, the cellulase activity in the recovered enzyme increases. In addition, it was found that degradation activities exhibit trends similar to those in cases where an inorganic salt of the reagent, such as sodium chloride, was used. However, it has been shown that Avicel's degradation activity is lower at pH 3.8 than at other pHs.
    (Example 17) Relationship between the addition of seawater and the temperature of secondary hydrolysis [0187] Secondary hydrolysis was carried out at temperatures of 30 ° C, 40 ° C, 50 ° C and 60 ° C in the presence of sea water at pH 5.0, which exhibited the highest addition effect among seawater, and the activity of the recovered enzyme was measured. Table 24 shows a summary of the
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    75/85 activity values represented as relative values calculated using as standard the activity of the enzyme recovered following secondary hydrolysis under the conditions of Example 1 (pretreated cellulose 3, 50 ° C) (activity = 1). Xylan degradation activity could not be detected for some conditions, and was represented as ND (not detected) in such cases. Since the xylan degradation activity was also ND for Example 1 (cellulose, pretreated product 3, 50 ° C), which was used as the standard, the xylan degradation activity observed in the case of sea water at 60 ° C was set to 1 to describe the relative activities.
    Table 24
    ActivityConditions Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan degradation activity Example 1 1 (Default) 1 (Default) 1 (Default) ND 30 ° C 4.4 3.0 2.0 3 40 ° C 7.4 3.3 2.1 3.2 50 ° C (Example16) 18 3.3 3.0 3 60 ° C 2.8 3.0 2.1 1 (Default)
    [0188] As shown in Table 24, it was found that, in the presence of sea water, especially the degradation activity of Avicel tends to increase as the temperature approaches 50 ° C, but the activity decreases at 60 ° C . In terms of CMC degradation activity and cellobiose degradation activity, no temperature dependent increase in the amount of recovery could be observed, and the degradation activities did not decrease even at a temperature above 60 ° C. In terms of the xylan degradation activity, it was found that the degradation activity of the recovered enzyme is higher at temperatures ranging from 30 ° C to 50 ° C,
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    76/85 but the activity is drastically reduced by 60 ° C. These trends were similar to those seen in Example 15 where sodium chloride was used.
    (Example 18) Relationship between Amino Acid Addition Amount in Secondary Hydrolysis and Amount of Enzyme Recovered [0189] As compounds to be added to secondary hydrolysis, several amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid , glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) were used. Secondary hydrolysis was carried out as in Example 1 (pretreated cellulose 3, 50 ° C), except that each of the amino acids was added in a final concentration of 1%. Aspartic acid and tyrosine could not be dissolved at the predetermined concentration and, therefore, the final concentration of 1% could not be reached. The pH after the addition was adjusted with hydrochloric acid and / or sodium hydroxide to a value in the range of 5.5 to 6.5. The experiment was carried out in the same way as in Example 1, except for the addition of the amino acid. Table 25 shows a summary of the activity values represented as relative values calculated using as standard the activity of the enzyme recovered after secondary hydrolysis under the conditions of Example 1 (cellulose, pretreated product 3, 50 ° C) (activity = 1). Xylan degradation activity could not be detected for some conditions, and was represented as ND (not detected) in such cases. As the xylan degradation activity was also ND for Example 1 (cellulose, pretreated product 3, 50 ° C), which was used as the standard, the detectable xylan degradation activity observed using lysine was defined as 1 to describe the relative activity for histidine.
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    77/85
    Table 25
    ^ '' „ActivityConditions·'-'-.. pH Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan degradation activity Example 1 5.0 1 (Default) 1 (Default) 1 (Default) ND Alanine 6.2 1.2 1.1 1.1 ND Arginine 5.6 3.7 1.3 1.3 ND Asparagine 5.5 1.3 1.1 1.1 ND Aspartic acid - Not feasible Not feasible Not feasible Not feasible Cysteine 6.5 7.4 1.4 1.4 ND Glutamine 5.4 1.2 1.1 1.1 ND Glutamic acid 6.0 1.7 1.1 1.1 ND Glycine 6.0 1.2 1.1 1.1 ND Isoleucine 6.0 3.1 1.2 1.2 1 Isoleucine 6.0 1.2 1.1 1.1 ND Leucine 6.0 1.2 1.1 1.1 ND Lysine 5.6 3.8 1.3 1.3 1 (Default) Methionine 5.8 1.2 1.1 1.1 ND Phenylalanine 5.8 1.2 1.1 1.1 ND Proline 6.1 1.2 1.1 1.1 ND Serina 5.8 1.2 1.1 1.1 ND Threonine 5.7 1.2 1.1 1.1 ND Tryptophan 5.7 1.2 1.1 1.1 ND Tyrosine - Not feasible Not feasible Not feasible Not feasible Valina 5.7 1.2 1.1 1.1 ND
    [0190] Consequently, as shown in Table 25, it was found that the addition of the amino acids tends to increase the amount of the enzyme recovered compared to the case in which no amino acid was added. Additionally, it was found that, especially by adding glutamic acid, lysine, histidine, arginine or cysteine among the amino acids, the
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    78/85 activity of the recovered enzyme can be increased. It was possible to confirm that the effect of adding cysteine is especially high in terms of Avicel's degradation activity. In addition, it was possible to confirm that the xylan degradation activity can be increased especially by the addition of lysine or histidine.
    (Example 19) Relationship between Amino Acid Addition and Secondary Hydrolysis Temperature [0191] Secondary hydrolysis was carried out at temperatures of 30 ° C, 40 ° C, 50 ° C and 60 ° C in the presence of 1% cysteine, which exhibited the highest addition effect among amino acids, and the activity of the recovered enzyme was measured. Table 26 shows a summary of the activity values represented as relative values calculated using as standard the activity of the enzyme recovered after secondary hydrolysis under the conditions of Example 1 (pretreated cellulose 3, 50 ° C) (activity = 1) . Xylan degradation activity could not be detected for any of the conditions, and represented as ND (not detected).
    Table 26
    ^ '' ' Activity Conditions' '' '' '' , ^ Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan degradation activity Example 1 1 (Default) 1 (Default) 1 (Default) ND 30 ° C 3.4 1.2 1.2 ND 40 ° C 6.8 1.2 1.4 ND 50 ° C (Example 18) 7.4 1.4 1.4 ND 60 ° C 1.5 1 1 ND
    [0192] As shown in Table 26, it was found that, in the presence of cysteine, especially the degradation activity of Avicel tends to increase as the temperature approaches 50 ° C, but the activity
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    79/85 decreases at 60 ° C. In terms of CMC degradation activity and cellobiose degradation activity, no temperature dependent increase in the amount of recovery could be observed, and the degradation activities did not decrease even at a temperature above 60 ° C. In terms of the xylan degradation activity, the activity could not be detected for any of the temperatures.
    (Example 20) Relationship between Quantity of Addition of Hydrophilic Organic Solvent in Secondary Hydrolysis and Recovered Enzyme Activity [0193] As compounds to be added to secondary hydrolysis, hydrophilic organic solvents (methanol, ethanol, 1-propanol, isopropanol) were used , dimethyl sulfoxide, N, N-dimethylformamide, acetone, acetonitrile, ethylene glycol and glycerin). Secondary hydrolysis was carried out as in Example 1 (pretreated cellulose 3, 50 ° C), except that each of the hydrophilic organic solvents was added in a final concentration of 1%. The experiment was carried out in the same way as in Example 1, except for the addition of the hydrophilic organic solvent. Table 27 shows a summary of the activity values represented as relative values calculated using as standard the activity of the enzyme recovered after secondary hydrolysis under the conditions of Example 1 (pretreated cellulose 3, 50 ° C) (activity = 1) . Xylan degradation activity could not be detected for any of the conditions, and represented as ND (not detected).
    Table 27
    Activity Conditions pH Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan degradation activity Example 1 5.0 1 (Default) 1 (Default) 1 (Default) ND
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    Activity Conditions pH Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan degradation activity Methanol 5.0 1.9 1.1 1.1 ND Ethanol 5.0 1.9 1.1 1.1 ND 1-Propanol 5.0 1.9 1.1 1.1 ND Isopropanol 5.0 1.9 1.1 1.1 ND Dimethyl sulfoxide 5.0 1.9 1.1 1.1 ND N, N-Dimethylformamid a 5.0 1.9 1.1 1.1 ND Acetone 5.0 1.9 1.1 1.1 ND Acetonitrile 5.0 1.9 1.1 1.1 ND Ethylene glycol 5.0 1.9 1.1 1.1 ND Glycerin 5.0 1.9 1.1 1.1 ND
    [0194] Consequently, as shown in Table 27, it was found that the activity of the recovered enzyme, especially the degradation activity of Avicel, can be increased with the addition of a hydrophilic organic solvent.
    (Comparative Example 6) Effect of Addition of Hydrophobic Organic Solvent on Secondary Hydrolysis [0195] As compounds to be added to secondary hydrolysis, n-hexane, 1-butanol and 1-pentanol were used, and secondary hydrolysis was performed using a procedure same as in Example 19. However, the hydrophobic organic solvents were separated from the aqueous phase and recovery with the ultrafiltration membrane was difficult. Regardless of being able to recover the enzyme or not, it was found that
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    81/85 hydrophobic organic solvents were unsuitable compounds for addition to secondary hydrolysis due to the difficulty of handling.
    (Example 21) Relationship between the addition of the hydrophilic organic solvent and the temperature of the secondary hydrolysis [0196] The secondary hydrolysis was carried out at temperatures of 30 ° C, 40 ° C, 50 ° C (as in Example 20) and 60 ° C in the presence of 1% ethanol, which is one of the hydrophilic organic solvents used in Example 20, and the activity of the recovered enzyme was measured. Table 28 shows a summary of the activity values represented as relative values calculated using as standard the activity of the enzyme recovered after secondary hydrolysis under the conditions of Example 1 (pretreated cellulose 3, 50 ° C) (activity = 1) . Xylan degradation activity could not be detected for any of the conditions, and represented as ND (not detected).
    [Table 28]
    ^ - .. Activity Conditions' '·' - ^^ Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan degradation activity Example 1 1 (Default) 1 (Default) 1 (Default) ND 30 ° C 1.2 1.1 1.1 ND 40 ° C 1.7 1.1 1.1 ND 50r: (Example 20) 1.9 1.1 1.1 ND 60 ° C 0.6 1 1.1 ND.
    [0197] It has been found that, in the presence of ethanol, especially the degradation activity of Avicel tends to increase as the temperature approaches 50 ° C, but the activity decreases at 60 ° C. In terms of CMC degradation activity and cellobiose degradation activity, no temperature-dependent increase in the amount of recovery could be observed, and the degradation activities did not decrease even in
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    82/85 a temperature above 60 ° C. In terms of the xylan degradation activity, the activity could not be detected for any of the temperatures.
    (Comparative Example 7) Effect of Addition of Water-Soluble Polymer on Secondary Hydrolysis [0198] As compounds to be added to secondary hydrolysis, several water-soluble polymers were used. As water-soluble polymers, polyallylamine-HCl-3S (PAA-3S, Nitto Boseki Co., Ltd.), polyallylamine-HCl-10S (PAA-10S, Nitto Boseki Co., Ltd.), polyethylene glycol # 4000 were used (PEG # 4000, Nakalai Tesque), polyethylene glycol # 6000 (PEG # 6000, Nakalai Tesque), polyethylene glycol # 20,000 (PEG # 20,000, Wako Pure Chemical Industries, Ltd.), polyvinyl alcohol 500 (PVA, Wako Pure Chemical Industries , Ltd.) and polyvinyl pyrrolidone (PVP, Sigma-Aldrich). Secondary hydrolysis was carried out as in Example 1 (cellulose, pretreated product 3, 50 ° C), except that each of the water-soluble polymers was added in a final concentration of 1%. The pH after the addition was adjusted with hydrochloric acid and / or sodium hydroxide to a value in the range of 5.5 to 6.5. The experiment was carried out in the same way as in Example 1, except for the addition of the water-soluble polymer. Table 29 shows a summary of the activity values represented as relative values calculated using as a standard the activity of the enzyme recovered after secondary hydrolysis under the conditions of Example 1 (cellulose, pretreated product 3, 50 ° C) (50 ° C) (activity = 1). Xylan degradation activity could not be detected for some conditions, and was represented as ND (not detected) in such cases.
    Table 29
    ^ -.... ActivityConditions '' · '' - ^ Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan degradation activity Example 1 1 (Default) 1 (Default) 1 (Default) ND
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    ^ '---... ActivityConditions '' '··' · ^^ Avicel degradation activity CMC degradation activity Cellobiose degradation activity Xylan degradation activity PAA-3S ND ND ND ND PAA-10S ND ND ND ND PEG # 4000 0.4 1 1 ND PEG # 6000 0.6 1 1 ND PEG # 20,000 0.2 0.5 1 ND PVA 0.6 1 1 ND PVP 0.5 1 1 ND
    [0199] In the presence of water-soluble polymers, no increase in the activity of the enzyme recovered due to its addition could be observed for any of the degradation activities.
    Industrial Applicability [0200] The sugar liquid obtained by the present invention can be used as a sugar material for various fermentation products.
    SYMBOLS DESCRIPTION
    Thermostat
    Agitation tank
    Cellulose inlet
    Shaking device
    Water supply line
    Warm water supply tank
    Warm water supply tank thermostat
    Filtration device using filter press
    Compressor
    Circulation line
    Filtrate recovery tank
    Ultrafiltration membrane device
    Carbohydrate recovery line
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    Hydrolyzate inlet
    Inlet of warm water
    External frame
    Filter cloth
    Solids (primary hydrolyzate)
    Pressing plate
    Interior of the filtration chamber using filter press
    Warm water inlet in conjunction with hydrolyzate inlet
    Sugar solution tank
    Nanofiltration membrane device or reverse osmosis membrane device
    Filtrate line
    Solid-liquid separation device
    Solid transfer medium
    Thermostat 2 (secondary hydrolysis tank)
    Secondary hydrolysis tank
    Stirring device 2 (secondary hydrolysis tank)
    Solid-liquid separation device 2 (secondary hydrolyzate)
    Secondary sugar liquid tank
    Secondary sugar liquid recovery tank
    Secondary sugar liquid ultrafiltration membrane device
    Secondary sugar liquid transfer line
    Secondary hydrolyzate transfer line
    Microfiltration membrane device
    Microfiltration membrane crude liquid tank
    Petition 870190015245, of 02/14/2019, p. 98/107
    85/85
    Microfiltration membrane
    Compressed air supply device
    Inverted washing pump
    Microfiltrate recovery tank
    权利要求:
    Claims (10)
    [1]
    Claims
    1. METHOD TO PRODUCE A SUGAR LIQUID, using cellulase from a filamentous fungus, characterized by understanding the steps of:
    (a) adding cellulase from a filamentous fungus to the cellulose and forming a primary hydrolyzate, and (b) then subjecting the primary hydrolyzate from step (a) to solid-liquid separation in a liquid of primary sugar and solids;
    (c) adding water to said solids from step (b) without further addition of enzyme and performing secondary hydrolysis on said solids to form a secondary hydrolyzate, (d) followed by subjecting the secondary hydrolyzate from step (c) to solid-liquid separation in a secondary sugar liquid and a residue; and (e) filtering said primary sugar liquid from step (b) and / or secondary sugar liquid from step (d) through an ultrafiltration membrane, and (f) recovering said cellulase from the feed side and recovering a liquid from permeate-side sugar, where the filamentous fungus cellulase is an enzyme composition comprising cellobiohydrolase, endoglucanase, exoglucanase, β-glucosidase, xylanase and xilosidase.
    [2]
    2. METHOD, according to claim 1, characterized in that said filamentous fungus cellulase is Trichoderma cellulase.
    [3]
    METHOD according to any one of claims 1 to 2, characterized in that said cellulose is derived from a pre-treated cellulose prepared by treatment with ammonia, treatment
    Petition 870190015245, of 02/14/2019, p. 100/107
    2/3 hydrothermal or biomass treatment with diluted sulfuric acid.
    [4]
    METHOD according to any one of claims 1 to 3, characterized in that said secondary hydrolysis is hydrolysis in the presence of one or more compounds selected from the group consisting of inorganic salts, except calcium salts, hydrophilic organic solvents, amino acids and non-ionic surfactants, and sugar liquids that comprise such substances.
    [5]
    5. METHOD according to claim 4, characterized in that said inorganic salt (s) is (are) one or more selected from the group consisting of sodium salts, potassium salts , magnesium salts, sulfuric acid salts, ammonium salts, hydrochloric acid salts, phosphoric acid salts, acetic acid salts and nitric acid salts, except calcium salts.
    [6]
    6. METHOD, according to claim 5, characterized by the said inorganic salt (s) serving one or more selected from the group consisting of sodium chloride, sodium acetate , sodium sulfate, sodium hydrogen sulfate, sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium chloride, ammonium chloride, dipotassium hydrogen phosphate, ammonium sulphate, magnesium chloride and magnesium sulphate.
    [7]
    METHOD according to claim 4, characterized in that said hydrophilic organic solvent (s) is one or more selected from the group consisting of methanol, ethanol , 1-propanol, isopropanol, N, N-dimethylformamide, butanol, acetone, acetonitrile, ethylene glycol and glycerin.
    [8]
    8. METHOD according to claim 4, characterized in that said amino acid (s) is one or more selected from the group consisting of arginine, cysteine, acid
    Petition 870190015245, of 02/14/2019, p. 101/107
    3/3 glutamic, histidine and lysine.
    [9]
    METHOD according to any one of claims 1 to 8, characterized in that said solid-liquid separation of a primary hydrolyzate and / or secondary hydrolyzate is filtration using a filter press.
    [10]
    10. METHOD, according to any one of claims 1 to 9, characterized in that it additionally comprises the step of:
    (g) filtering said sugar liquid from step (f) through a reverse osmosis membrane and / or nanofiltration membrane to concentrate said sugar liquid.
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    同族专利:
    公开号 | 公开日
    RU2012143744A|2014-04-20|
    US20130059345A1|2013-03-07|
    MY180119A|2020-11-23|
    WO2011115039A1|2011-09-22|
    EP2548965A4|2015-12-16|
    KR20130016240A|2013-02-14|
    AU2011228212A1|2012-10-25|
    AU2011228212B2|2015-03-26|
    BR112012023159A2|2015-10-06|
    CA2792089A1|2011-09-22|
    JP6003056B2|2016-10-05|
    DK2548965T3|2018-11-19|
    US20160010046A1|2016-01-14|
    ES2687821T3|2018-10-29|
    CA2792089C|2018-05-01|
    RU2560443C2|2015-08-20|
    KR101778111B1|2017-09-13|
    US9150895B2|2015-10-06|
    EP2548965A1|2013-01-23|
    JPWO2011115039A1|2013-06-27|
    EP2548965B1|2018-08-22|
    CN102791873A|2012-11-21|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US3642580A|1970-01-08|1972-02-15|Us Army|Enzymatic saccharification of cellulose|
    US3764475A|1971-12-22|1973-10-09|Us Army|Enzymatic hydrolysis of cellulose to soluble sugars|
    US4220721A|1979-04-27|1980-09-02|University Of Arkansas Foundation|Method for enzyme reutilization|
    SU949002A1|1980-11-27|1982-08-07|Московский Орденов Ленина,Октябрьской Революции И Трудового Красного Знамени Государственный Университет Им.М.В.Ломоносова|Process for producing sugar from cellulose-containing raw material|
    JPS6024713B2|1983-03-18|1985-06-14|Kogyo Gijutsuin|
    US4713334A|1983-03-18|1987-12-15|Agency Of Industrial Science And Technology|Process for the saccharification of celluloses|
    JPS619039B2|1983-05-20|1986-03-19|Kogyo Gijutsuin|
    JPH0341380B2|1984-08-23|1991-06-21|
    JPS6387994A|1986-10-02|1988-04-19|Res Assoc Petroleum Alternat Dev<Rapad>|Production of sugar syrup and enzyme from saccharified liquid|
    JPS63137691A|1986-11-28|1988-06-09|Kobe Steel Ltd|Recovery of saccharifying enzyme|
    RU2030892C1|1992-09-22|1995-03-20|Всероссийский научно-исследовательский институт консервной и овощесушильной промышленности|Vegetable raw material reprocessing line|
    EP0832276B1|1995-06-07|2005-03-02|Arkenol, Inc.|Method of strong acid hydrolysis|
    JPH07231776A|1994-02-24|1995-09-05|Kirin Eng Kk|Apparatus for producing fermented sugar|
    JP3041380B2|1997-06-02|2000-05-15|工業技術院長|Process for producing water-soluble oligosaccharides and monosaccharides|
    JP2001095597A|1999-07-27|2001-04-10|Shiseido Co Ltd|Detection of steroid 5 alpha-reductase-inhibiting activity|
    JP4330839B2|2002-01-18|2009-09-16|旭化成ケミカルズ株式会社|Method for producing glucose and / or water-soluble cellooligosaccharide|
    US7189306B2|2002-02-22|2007-03-13|Gervais Gibson W|Process of treating lignocellulosic material to produce bio-ethanol|
    CN1216150C|2002-04-12|2005-08-24|中国农业大学|Method for producing alcohol by solid fermentation of stalks|
    JP2005229821A|2004-02-17|2005-09-02|Jgc Corp|Method for producing monosaccharide from biomass and apparatus for producing monosaccharide|
    JP4554314B2|2004-09-22|2010-09-29|財団法人地球環境産業技術研究機構|Continuous saccharification method of lignocellulose|
    EP1869197A2|2005-04-12|2007-12-26|E.I. Dupont De Nemours And Company|Treatment of biomass to obtain ethanol|
    CN1944658B|2006-06-09|2010-07-14|山东大学|Method for producing cellulose alcohol using corncob processing leftover by fermenting|
    CA2655640A1|2006-06-22|2007-12-27|Iogen Energy Corporation|Enzyme compositions and methods for the improved enzymatic hydrolysis of cellulose|
    JP5109121B2|2006-12-28|2012-12-26|国立大学法人東京大学|Method for producing sugar, method for producing ethanol, method for producing lactic acid, cellulose for enzymatic saccharification used therein and method for producing the same|
    JP2008206484A|2007-02-28|2008-09-11|Oji Paper Co Ltd|Method for collecting enzyme|
    CN101285106B|2008-06-10|2010-08-18|南京工业大学|Process for preparing multicomponent liquid glucose and lignose while effectively hydrolyzing lignocellulosic biomass|
    CN101434977A|2008-12-23|2009-05-20|中国石油化工股份有限公司|Novel method for saccharification of ligno-cellulose|
    JP2011019483A|2009-07-17|2011-02-03|Jgc Corp|Saccharified solution preparation method and saccharification reaction device|JPH0919301A|1995-07-04|1997-01-21|Bangaade:Kk|Portable slippers|
    US10980244B2|2008-11-04|2021-04-20|The Quaker Oats Company|Whole grain composition comprising hydrolyzed starch|
    US10689678B2|2008-11-04|2020-06-23|The Quaker Oats Company|Method and composition comprising hydrolyzed starch|
    JP5621528B2|2010-11-12|2014-11-12|王子ホールディングス株式会社|Enzymatic saccharification method of lignocellulosic material|
    EP2688421B1|2011-03-21|2015-12-30|PepsiCo, Inc.|Method for preparing high acid rtd whole grain beverages|
    US10179924B2|2011-03-29|2019-01-15|Toray Industries, Inc.|Method for producing sugar solution|
    CN103796523B|2011-07-12|2016-01-27|百事可乐公司|Preparation is containing the method for oats milk beverage|
    US9499783B2|2011-09-14|2016-11-22|Toray Industries, Inc.|Production apparatus of sugar solution and production system of sugar solution|
    IN2014CN03792A|2011-11-21|2015-10-16|Toray Industries|
    US10519476B2|2012-05-18|2019-12-31|Toray Industries, Inc.|Method of producing sugar liquid|
    UA118174C2|2012-07-02|2018-12-10|Ксілеко, Інк.|METHOD OF BIOMASS PROCESSING|
    BR112014032455A2|2012-07-03|2017-06-27|Toray Industries|method for producing a sugar liquid|
    NZ706072A|2013-03-08|2018-12-21|Xyleco Inc|Equipment protecting enclosures|
    JP2014100073A|2012-11-16|2014-06-05|Toray Ind Inc|Composition for biomass degradation and production process for sugar solution using it|
    FR2999605A1|2012-12-14|2014-06-20|IFP Energies Nouvelles|PROCESS FOR PRODUCING SUGAR SOLUTIONS FROM LIGNOCELLULOSIC BIOMASS WITH COMPLEMENTARY TREATMENT OF THE SOLID RESIDUE BY A HYDRATED INORGANIC SALT|
    JP2014128213A|2012-12-28|2014-07-10|Kawasaki Heavy Ind Ltd|Method of producing concentrated saccharified liquid|
    JP6307789B2|2013-01-07|2018-04-11|東レ株式会社|Sugar solution manufacturing apparatus and sugar solution manufacturing method|
    CA2901967C|2013-02-20|2020-11-17|Chiaki Yamada|Sugar-solution production method|
    WO2014208493A1|2013-06-25|2014-12-31|東レ株式会社|Method for producing sugar solution|
    MY172577A|2013-07-12|2019-12-03|Toray Industries|Method for producing alcohol from cellulose-containing biomass|
    TW201522380A|2013-10-02|2015-06-16|Basf Se|Method for processing cellulose-containing biomass|
    BR112016014736A2|2013-12-27|2020-09-08|Toray Industries, Inc.|METHOD FOR THE PRODUCTION OF A SUGAR LIQUID|
    US10279316B2|2014-05-08|2019-05-07|Thetis Environmental Inc.|Closed loop membrane filtration system and filtration device|
    WO2016047589A1|2014-09-26|2016-03-31|東レ株式会社|Method of preparing sugar solution|
    KR20180012254A|2015-04-10|2018-02-05|코멧 바이오리파이닝 인코포레이티드|Methods and compositions for the treatment of cellulosic biomass and products produced thereby|
    EP3098320A1|2015-05-29|2016-11-30|Clariant International Ltd|Process for the hydrolysis of biomass|
    WO2016207144A1|2015-06-22|2016-12-29|Dsm Ip Assets B.V.|Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars|
    WO2016207146A1|2015-06-22|2016-12-29|Dsm Ip Assets B.V.|Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars|
    US20170275662A1|2016-03-22|2017-09-28|The Quaker Oats Company|Method and Apparatus for Controlled Hydrolysis|
    US11172695B2|2016-03-22|2021-11-16|The Quaker Oats Company|Method, apparatus, and product providing hydrolyzed starch and fiber|
    FI20165250A|2016-03-24|2017-09-25|Upm-Kymmene Corp|Method and apparatus for enzymatic hydrolysis as well as liquid fraction and solid fraction|
    FI20165466A|2016-06-03|2017-12-04|Upm-Kymmene Corp|Method and apparatus for enzymatic hydrolysis, liquid fraction and solid fraction|
    JP6513060B2|2016-08-04|2019-05-15|ユニ・チャーム株式会社|Method of producing saccharified liquid from used absorbent article|
    CN106092894B|2016-08-23|2018-07-13|兆光生物工程有限公司|A kind of membrane filtration separation detecting device|
    CN106222314B|2016-08-31|2020-04-03|中国科学院广州能源研究所|Method for hydrolyzing biomass in two steps by using carbon-based solid acid catalyst|
    FI20165782A|2016-10-13|2018-04-14|Upm Kymmene Corp|Process and equipment for enzymatic hydrolysis, liquid fraction and lignin fraction|
    RU2630452C1|2016-11-01|2017-09-08|Федеральное государственное бюджетное образовательное учреждение высшего образования "Воронежский государственный университет инженерных технологий" |Method for pre-treatment of sugar beet chips when obtaining diffusion juice|
    EP3530743A1|2018-02-21|2019-08-28|Cambridge Glycoscience Ltd|Method of production|
    JPWO2019189651A1|2018-03-29|2021-02-12|東レ株式会社|Method for producing purified sugar solution|
    EP3790409A4|2018-05-10|2021-07-21|Comet Biorefining Inc.|Compositions comprising glucose and hemicellulose and their use|
    BR112021002910A2|2018-08-15|2021-07-20|Cambridge Glycoscience Ltd|new compositions, their use and methods for their formation|
    CN109430816A|2018-11-17|2019-03-08|湖南犟哥生态农业有限公司|A kind of pueraria starch processing slag and effluent processing method and equipment|
    CN110772995A|2019-10-24|2020-02-11|蔡林|Barley β -amylase extraction process|
    WO2021235419A1|2020-05-19|2021-11-25|東レ株式会社|Method for producing proteins using corn hulls|
    法律状态:
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2018-11-21| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2019-03-06| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2019-05-07| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/03/2011, OBSERVADAS AS CONDICOES LEGAIS. (CO) 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/03/2011, OBSERVADAS AS CONDICOES LEGAIS |
    优先权:
    申请号 | 申请日 | 专利标题
    JP2010-057403|2010-03-15|
    JP2010057403|2010-03-15|
    PCT/JP2011/055902|WO2011115039A1|2010-03-15|2011-03-14|Manufacturing method for sugar solution and device for same|
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